Mouse Modeling Dissecting Macrophage–Breast Cancer Communication Uncovered Roles of PYK2 in Macrophage Recruitment and Breast Tumorigenesis

Abstract Macrophage infiltration in mammary tumors is associated with enhanced tumor progression, metastasis, and poor clinical outcome, and considered as target for therapeutic intervention. By using different genetic mouse models, the authors show that ablation of the tyrosine kinase PYK2, either in breast cancer cells, only in the tumor microenvironment, or in both, markedly reduces the number of infiltrating tumor macrophages and concomitantly inhibits tumor angiogenesis and tumor growth. Strikingly, PYK2 ablation only in macrophages is sufficient to induce similar effects. These phenotypic changes are associated with reduced monocyte recruitment and a substantial decrease in tumor‐associated macrophages (TAMs). Mechanistically, the authors show that PYK2 mediates mutual communication between breast cancer cells and macrophages through critical effects on key receptor signaling. Specifically, PYK2 ablation inhibits Notch1 signaling and consequently reduces CCL2 secretion by breast cancer cells, and concurrently reduces the levels of CCR2, CXCR4, IL‐4Rα, and Stat6 activation in macrophages. These bidirectional effects modulate monocyte recruitment, macrophage polarization, and tumor angiogenesis. The expression of PYK2 is correlated with infiltrated macrophages in breast cancer patients, and its effects on macrophage infiltration and pro‐tumorigenic phenotype suggest that PYK2 targeting can be utilized as an effective strategy to modulate TAMs and possibly sensitize breast cancer to immunotherapy.


Introduction
TAMs, the most abundant immune cells in the tumor microenvironments (TME), originate from circulating blood monocytes and tissue resident macrophages. [1,2] Monocytes are initially recruited into the TME by multiple chemokines (CCL2, CSF-1, CXCL12) secreted by malignant and stromal cells, and subsequently differentiate locally into TAMs. [2] In breast cancer, and particularly in triple negative breast cancer (TNBC), infiltrated TAMs facilitate tumor growth, enhance angiogenesis, immunosuppression, and drug resistance, and are highly correlated with poor prognosis. [3,4] These protumorigenic effects are thought to be mediated by mutual communication between breast cancer cells, TAMs, and other cells of the TME. [5] A wide spectrum of TAMs displaying heterogeneous phenotypes has been identified in the TME. [6] Although oversimplified, TAMs have been classified into two extreme phenotypes: classically activated (M1) and alternatively activated (M2) macrophages. Inflammatory anti-tumorigenic M1-like macrophages are typically activated by Th1 cytokines, such as IFN-and CSF-2 or by lipopolysaccharide. These cells are characterized by secretion of pro-inflammatory cytokines, including TNF-, IFN-, IL-12, and IL-23, [7] by high expression of nitric oxide synthase 2 (NOS2) and production of NO or reactive oxygen species, and by promotion of Th1 lymphocyte development. [8] By contrast, immunosuppressive protumorigenic M2-like macrophages are activated by Th2 cytokines including IL-4, IL-13, and IL-10, [9] which leads to activation of several transcription factors including STAT6, upregulation of certain chemokines (such as CCL17, CCL22, and CCL24) and anti-inflammatory cytokines (such as IL-10, TGF-), which promote the development of Th2 lymphocytes and regulatory T cells (Tregs). M2-like macrophages express specific cellular markers, including ARG1, YM1, FIZZ1, and the cell-surface scavenger receptors MRC1/CD206 and CD163. [6,10,11] These cells support tumor progression and metastasis through several mechanisms, including secretion of numerous growth factors, cytokines, and ECM-remodeling molecules and metalloproteinases that promote tumor growth, migration, and angiogenesis. [12] Despite the characteristic features of M1/M2-like macrophages, these two extreme activation states display phenotypic plasticity and can switch in response to microenvironmental cues (inflammation, pathogens, tumor cell death) from anti-inflammatory/pro-tumorigenic (M2-like) toward pro-inflammatory/anti-tumorigenic (M1-like), which suppress tumor growth. [7] M1-and M2-like TAMs share characteristic features with MHCII high and MHCII low TAMs, respectively. MHCII high TAMs, which usually appear at early stages of tumor development, display tumor-suppressive activity, while MHCII low TAMs are more dominant at progression phases and exhibit tumor-promoting effects. [13] Increasing evidence suggests that pro-tumorigenic TAMs are predominant in the TME of most cancers. [6] Therefore, targeting their survival/proliferation, recruitment, and/or polarization into an M2-like pro-tumorigenic phenotype have been proposed as effective strategies for breast cancer therapy, possibly in combination with other therapeutic agents like chemotherapy or immune checkpoint blockade (ICB). [14,15] We previously showed that co-targeting of the non-receptor tyrosine kinase PYK2 and EGFR could be beneficial for basallike breast cancer with high-to-moderate expression levels of EGFR. [16,17] In addition, PYK2 expression exhibits significant correlation with breast cancer grade and lymph node metastasis. [18,19] PYK2 is also highly expressed in macrophages and affects macrophage infiltration into inflammatory regions, macrophage motility and phagocytosis, as well as macrophagemediated inflammatory responses. [20][21][22] Previous studies have shown that coinhibition of PYK2 together with its closely related kinase FAK using a dual-kinase inhibitor reduced TAMs in xenograft models of breast cancer, [23] and recent studies suggest that activation of PYK2 by tumor cell-derived spondin 2 (SPON2), an extracellular matrix glycoprotein that binds and activates integrin 1 signaling, is essential for macrophage infiltration in colorectal cancer. [24] These studies imply that PYK2 may also play a role in breast cancer-macrophage communication. However, the influence of PYK2 on TAMs has not been thoroughly defined, and currently, a highly potent and selective PYK2 inhibitor is not available. Furthermore, a systemic admin-istration of pharmacological drug simultaneously affects both the tumor cells and the TME, and thus cannot be used to dissect the discrete contribution of each compartment for tumor development. In this study, we applied genetic approaches to ablate the PYK2 gene (PTK2B) either in breast cancer cells using the CRISPR/Cas9 technology, in the TME (total knockout mice), or selectively in macrophages (Cx3cr1-Cre tg/wt /PYK2 f/f mice). These genetic models enabled us to selectively dissect the functional impact of PYK2 in macrophages, tumor cells, and in macrophagetumor cell communication, and to uncover key pathways that are regulated by PYK2 and modulate TAM recruitment and polarization.

PYK2 Modulates TAM Infiltration and Breast Cancer Growth
To define the influence of PYK2 on breast cancer growth, we established a syngeneic breast cancer model by orthotopic implantation of wild-type (WT) and PYK2 knockout (KO) breast cancer (BC) cells into WT or PYK2 ko/ko (PYK2 KO) C57BL/6 mice (Figure 1A,B), [25,26] respectively. In the absence of a selective PYK2 inhibitor, these mouse models recapitulate systemic drug administration in an immunocompetent microenvironment. The breast cancer cell line EO771, derived from a spontaneous mammary tumor in C57BL/6 mice, [27,28] was used to establish PYK2 KO cell lines by CRISPR/Cas9 technology (Experimental Section). Several EO771 clones were generated ( Figure S1A, Supporting Information) and two independent clones (KO2 and KO12) of two distinct sgRNAs were selected for further analysis ( Figure 1C). Control (WT) and PYK2 KO EO771 cells (KO2) were implanted into the mammary fat pad of WT and PYK2 KO mice. The absence of PYK2 in breast tissue of PYK2 KO mice was confirmed by Western blotting ( Figure 1D).
Tumor growth was measured over time with calipers until reaching a volume of ≈0.6 cm 3 ( Figure 1E). As shown in Figure 1E, implantation of PYK2 KO BC cells into the mammary fat pad of PYK2 KO mice significantly attenuated tumor growth compared to WT control, and significantly reduced tumor volume (by ≈40%, p = 0.004) at the endpoint (day 20). Hematoxylin and Eosin (H&E) staining of tumor sections (Figure S1B, Supporting Information) revealed a less condensed tumor tissue in the PYK2 KO compared to WT control, and immunohistochemistry (IHC) analysis for PYK2 confirmed the ablated genotype in the tumor sections ( Figure S1C, Supporting Information). Importantly, despite the reduced tumor growth of implanted PYK2 KO EO771 cells (KO2, KO12), these cells had the same proliferation rate as WT cells in vitro ( Figure S1D, Supporting Information), implying that the TME of PYK2 KO mice imposes tumor inhibitory effects.
It is well known that PYK2 is expressed in different cell types of the TME, [29] and our immunofluorescence (IF) analysis confirmed its high protein expression in various immune cells (T cells, NK cells, monocytes), moderate expression in endothelial cells and a very low expression in fibroblasts ( Figure S1E-I, Supporting Information). Since PYK2 ablation had profound effects on macrophages, [20] and TAMs are the major component of the TME that strongly contribute to tumor development and progression, [30] we examined the influence of PYK2 KO . E) Tumor growth curves over 20 days show the mean tumor volume at the indicated time points following EO771 implantation (WT and KO) in WT mice (n = 10) or PYK2 KO mice (n = 11); statistical significance was determined by mixed-effects model. Volume of individual tumor at day 20 is shown in the box plot; statistical significance was determined by Welch t-test. F) Quantification of F4/80-positive cells in tumor sections from WT (n = 9) and PYK2 KO (n = 6) mice at day 20 following implantation (Experimental Section). Statistical significance was determined by Welch t-test. Representative immunohistochemical images are shown. Scale bar: 50 μm. BC (breast cancer). on macrophage infiltration. We used the murine macrophage marker F4/80 and IHC analysis. As seen in Figure 1F, the number of F4/80-positive macrophages was substantially reduced in PYK2 KO tumors compared to the WT. These results suggest that the inhibitory effects of PYK2 ablation on tumor growth were mediated, at least in part, by reduced number of TAMs and their pro-tumorigenic effects.

Dissecting the Distinctive Impact of PYK2 Deficiency in Breast Cancer Cells and the TME
To explore the possibility that PYK2 KO reduces TAM numbers, thereby suppressing their pro-tumorigenic effects and attenuates BC growth, we systematically dissected the impact of PYK2 ablation either in the BC cells or in the TME using different mouse models. First, we evaluated the impact of PYK2 ablation only in BC cells by comparing tumor growth of WT and PYK2 KO EO771 cells implanted in WT mice (Figure 2A). As shown in Figure 2B and Figure S2A (Supporting Information), PYK2 ablation in BC cells (two independent clones of two sgRNAs; KO2, Figure 2B, and KO12, Figure S2A, Supporting Information) also reduced tumor growth by ≈40%, and the excised tumors displayed similar H&E staining of less condensed tissue (Figure S2B, Supporting Information). Importantly, the inhibitory effects on tumor growth were associated with a significantly reduced number of infiltrating macrophages as quantified by IHC analysis for F4/80 ( Figure 2C; Figure S2C, Supporting Information). To confirm the IHC results on infiltrated macrophages, we performed flow cytometry analysis of size-matched tumors ( Figure S2D, Supporting Information). We adapted a protocol described by Bolli et al., [31] and defined macrophages by their CD45 + CD11b + Ly6C lo-int Ly6G − SiglecF − F4/80 hi phenotype ( Figure S2F, Supporting Information). As shown in Figure 2D, a profound reduction of total macrophages was obtained in PYK2 KO tumors as well as significantly less Ly6C hi monocytes (Figure 2E), which may represent a subset of myeloid-derived suppressor cells. [32] In a second model, where WT BC cells (EO771) were implanted into PYK2 KO mice ( Figure 2F), we also observed a significant reduction in tumor volume (≈50% at the endpoint, p = 0.0004) compared to WT BC cells grown in WT background ( Figure 2G), thus highlighting the impact of PYK2 ablation only within the TME. The inhibitory effects on tumor growth were accompanied by a massive decrease in the number of infiltrated macrophages as shown by F4/80 immunostaining ( Figure 2H). Flow cytometry analysis of sizematched tumors ( Figure S2E, Supporting Information) supported the IHC results and showed a substantial reduction in CD45 + CD11b + Ly6C lo-int Ly6G − SiglecF − F4/80 hi macrophages ( Figure 2I), while Ly6C hi monocyte numbers were unchanged ( Figure 2J).
Collectively, these results show that ablation of PYK2 either in BC cells or in the TME substantially reduced TAM numbers. A summary of the different mouse models and the effect of the respective PYK2 manipulations on both tumor size and macrophage infiltrates (IHC analysis) is shown in Figure 2K. These different models highlight the discrete impact of PYK2 ablation either in BC cells or the TME as well as the therapeutic potential of an effective and selective inhibitor for PYK2.

Ablation of PYK2 in Breast Cancer Cells Impairs CCL2 Secretion, Notch1 Signaling, and Macrophage Recruitment
The finding that PYK2 depletion in BC cells massively reduced the number of infiltrated macrophages ( Figure 2C,D) implies that PYK2 is required for monocyte/macrophage attraction. Since many secreted factors, including chemokines, cytokines, and growth factors can mediate the crosstalk between tumor cells and macrophages, [30] we first examined the influence of PYK2 depletion in BC cells on chemotaxis of macrophages in vitro using transwell assays. We examined both mouse and human breast cancer cell lines; the mouse system included both WT and PYK2 KO BC EO771 cells and the murine Raw264.7 macrophage line, while the human system included several TNBC cell lines (BT549, MDA-MB-231, Hs578T; WT and PYK2 knockdown (KD)) and the human monocytic cell line THP-1. [33] As shown in Fig-www.advancedsciencenews.com www.advancedscience.com ure 3A,B, depletion of PYK2 in BC cells significantly attenuated chemotaxis of macrophages toward tumor cells, suggesting that PYK2 depletion modulates the secretome of cancer cells and consequently macrophage attraction.
To identify the relevant secreted factors, we analyzed conditioned media of WT and PYK2-depleted human BT549 and MDA-MB-231 cells by cytokine array (80 Human Cytokines: AAH-CYT-G5; RayBiotech) ( Figure 3C; Figure S3A, Supporting Information). Several cytokines known to be involved in monocyte/macrophage recruitment were substantially reduced in the supernatants of the two PYK2-depleted TNBC cell lines, such as IL-1 and IL-6, while other chemokines (CCL2, CXCL7, CCL7, CCL15) were strongly reduced at least in BT549. [34][35][36][37][38] As CCL2 plays a major role in monocyte recruitment to most solid tumors, [34] we validated the results for this chemokine by ELISA using conditioned media of WT and PYK2-depleted human TNBC cell lines ( Figure 3D) as well as murine EO771 cells ( Figure 3E; Figure S3B, Supporting Information). We also analyzed tumor lysates derived from WT and PYK2 KO BC cells (EO771) in WT mice ( Figure 3F; Figure S3C, Supporting Information). As shown in Figure 3D-F, PYK2 depletion significantly reduced the levels of secreted CCL2 from human and mouse BC cell lines in vitro, as well as the level of CCL2 in breast tumor lysates. We then examined whether PYK2 depletion affects CCL2 transcription in human TNBC cell lines, murine EO771 BC cells, and EO771-derived tumor tissues (Figure 3G-I; Figure S3D,E, Supporting Information) by qPCR. Significant reduction was observed in all cases analyzed, indicating that PYK2 affects CCL2 transcription.
The influence of PYK2 on CCL2 transcription ( Figure 3G-I), led us to examine whether PYK2 regulates CCL2 transcription via Notch signaling, as previous studies showed that Notch1 regulates IL-1 and CCL2 expression in mammary carcinoma and consequently modulates macrophage recruitment. [39] We, therefore, examined the level of Notch1 protein in WT and PYK2-depleted human TNBC cell lines ( Figure 4A) or murine EO771 cells ( Figure 4B; Figure S4A, Supporting Information). As Notch1 activation requires proteolytic release of Notch1 intracellular domain (N1ICD) and its subsequent nuclear translocation to regulate transcription of target genes, [40,41] we assessed both full-length Notch1 and N1ICD by WB. As shown in Figure 4A,B and Figure S4A (Supporting Information), PYK2 depletion had no obvious effect on full-length Notch1, but substantially reduced N1ICD levels. Cell fractionation showed that the N1ICD was present both in the nucleus and the cytosol of WT EO771 cells, but only weakly detected in the cytosol of PYK2 KO cells ( Figure 4C; Figure S4B, Supporting Information). These results suggest that PYK2 regulates either the nuclear translocation of N1ICD and/or N1ICD stability. Indeed, treatment of PYK2 KO EO771 cells with the proteasome inhibitor MG132 restored the level of N1ICD ( Figure 4D; Figure S4C, Supporting Information), suggesting that PYK2 stabilizes N1ICD. Importantly, overexpression of PYK2 in PYK2 KO EO771 cells rescued N1ICD levels (Figure 4E), thus highlighting the specificity of PYK2 and excluding off-target effects.
To further corroborate the link between Notch1 and CCL2 in the BC cell lines, we inhibited N1ICD release by the gamma secretase inhibitor (GSI) LY411575. We confirmed the inhibition by Western blotting with an antibody against N1ICD ( Figure S4D, E, Supporting Information), and showed by ELISA that CCL2 secretion was reduced in both the human and murine BC cells ( Figure 4F,G). GSI treatment also reduced transcription of CCL2 ( Figure 4H), transcription of Notch1 itself and of the Notch target gene Hes1 ( Figure S4F, Supporting Information) in EO771 cells.
To directly demonstrate the influence of Notch1 on the Ccl2 transcript, we depleted Notch1 by shRNA in EO771 cells (Figure 4I) and examined Ccl2 mRNA levels by qPCR. As shown in Figure 4J, Notch1 knockdown markedly reduced Ccl2 level (by 60%) as well as Plau, another Notch1 regulated factor, [42] whereas Hes1, a common target gene of all Notch family members, was not changed, indicating that Hes1 is Notch dependent but Notch1-independent in these cells ( Figure S4G, Supporting Information). [43] Importantly, we found that PYK2 gene expression (PTK2B) is correlated with CCL2 ( Figure 4K; Figure  S4H, Supporting Information) as well as IL1B ( Figure S4I, Supporting Information) expression in breast cancer patients, further strengthening the link between PYK2 and these Notch1regulated cytokines and highlighting the clinical relevance of our findings.

Ablation of PYK2 Only in Macrophages Is Sufficient to Reduce Tumor Growth and Macrophage Infiltration
The remarkable effects of PYK2 ablation either in BC cells or in the TME on tumor growth and macrophage infiltration (Figure 2) highlight a link between PYK2 and TAMs. To demonstrate its clinical relevance, we assessed the correlation between PYK2 expression (PTK2B gene) and macrophage markers (CD68, CD163 [44] ) in human breast cancer datasets. As shown in To further characterize the functional link between PYK2 and TAMs, we examined the effects of PYK2 ablation only in macrophages on tumor growth and macrophage infiltration. To this end, we generated macrophage specific PYK2 KO mice (Cx3cr1-Cre/PYK2 f/f , referred to as Mϕ-KO) by crossing PYK2 f/f mice with mice expressing the Cre recombinase under control of the Cx3cr1 promotor. [45] First, we confirmed the KO of PYK2 in macrophages by Western blot analysis of bone marrow-derived macrophages (BMDMs) from Mϕ-KO compared to Cx3cr1-Cre tg/wt /PYK2 wt/wt (Mϕ-WT) mice ( Figure 5C). Then, WT EO771 cells were orthotopically implanted into the mammary fat pad of Mϕ-WT and Mϕ-KO mice ( Figure 5D) and tumor growth was measured over time. Tumor growth was significantly reduced in Mϕ-KO mice compared to Mϕ-WT mice with an average reduction of 38% after 24 days ( Figure 5E). No obvious microscopic changes were observed by H&E staining of tumor  sections ( Figure S5C, Supporting Information). IHC analysis revealed a significant reduction of F4/80 + macrophages in tumors from Mϕ-KO compared to Mϕ-WT mice ( Figure 5F).
Importantly, a decrease in tumor volume (≈34%, Figure S5E, Supporting Information) and number of infiltrated macrophages ( Figure S5F, Supporting Information) was also obtained when PYK2 KO EO771 cells were orthotopically implanted into Mϕ-KO mice ( Figure S5D, Supporting Information). Figure 5G summarizes tumor sizes and macrophage numbers (IHC) for the macrophage-specific PYK2 depletion system. The data highlight the crucial role of TAMs for breast cancer growth.

Defects of PYK2 Knockout Macrophages
As shown in Figure 5, selective ablation of PYK2 in macrophages significantly attenuated tumor growth and concomitantly reduced TAM numbers, implying that ablation of PYK2 in macrophages results in cell autonomous defects and/or impairs their crosstalk with tumor cells. To address these possibilities, we first examined the influence of PYK2 ablation in macrophages on cell migration by modified Boyden chamber migration assays. As shown in Figure 6A, migration of PYK2 KO Raw264.7 cells toward complete medium (10% fetal bovine serum (FBS)) was markedly reduced compared to WT Raw264.7 cells, suggesting a cell-autonomous effect of PYK2. In addition, we observed defects in migration of PYK2 KO Raw264.7 cells ( Figure 6B) as well as PYK2 KO BMDMs toward WT BC cells (EO771) (Figure 6C), suggesting effects of PYK2 on BC-macrophage communication.
To better characterize the phenotype of PYK2 depleted macrophages, we performed bulk RNA sequencing of BMDMs derived from WT and PYK2 KO mice. Gene expression of PYK2 KO BMDMs compared to WT control is shown in Table S1 of the Supporting Information, which confirmed the decrease in PTK2B expression ( Figure S6A, Supporting Information). Gene set enrichment analysis (GSEA) highlighted changes in key pathways including cell-cell communication, cell migration, chemotaxis, and several pathways related to macrophage function, including processing of external signals, macrophage immunity and antigen presentation, as well as the IL-4 pathway ( Figure 6D). These pathways of macrophage-autonomous defects are consistent with previous reports demonstrating impaired macrophage migration, cell polarization, [20] and phagocytic activity in PYK2 KO mice. [46] The RNA-seq data ( Figure 6D) together with the chemotaxis results ( Figure 6A-C) and our previous studies demonstrating the broad influence of PYK2 on different cell surface receptors [16,19,47] suggest that PYK2 may affect key chemotactic receptors as well as IL-4R . Hence, we first examined the effect of PYK2 KO on two major chemoattractant receptors in macrophages, CCR2 and CXCR4, [48] by WB. As shown, KO of PYK2 markedly reduced the protein levels of these two receptors in Raw264.7 cells (Figure 6E,F) and in BMDMs ( Figure 6G, H). Importantly, we also found reduced surface expression of CCR2 on BMDMs (Figure 6I) and of CXCR4 on TAMs ( Figure 6J) using flow cytom- etry analysis. We further showed that PYK2 depletion enhanced lysosomal degradation of these receptors, as lysosomal inhibitors (chloroquine, NH 4 Cl, bafilomycin (Baf1)) rather than the proteasomal inhibitor MG132 could restore the receptor levels (Figure 6K, L).
The reduced surface expression of CCR2 on BMDMs was accompanied with significantly reduced chemotaxis of PYK2 KO BMDMs toward media containing recombinant CCL2 in the lower chamber ( Figure 6M). Moreover, ex vivo analysis of monocytes enriched from BM (BMDMo) further confirmed that PYK2 depletion impaired migration toward CCL2 ( Figure 6N; Figure S6B, Supporting Information). Altogether, these findings demonstrate that PYK2 ablation reduced the levels of key chemotactic receptors and consequently of monocyte trafficking. These observations highlight the dual role of PYK2 in regulating CCL2-CCR2 signaling through concurrent influence on CCL2 release from breast cancer cells and of CCR2 protein levels in macrophages.

PYK2 Modulates IL-4R Signaling in Macrophages and Influences Their Pro-tumorigenic Phenotype
As mentioned, the RNA-seq analysis indicated that the IL-4 pathway was significantly altered in PYK2 KO BMDMs (Figures 6D and 7A). This pathway is critical for induction of a protumorigenic phenotype. [49] To validate the impact of PYK2 on this pathway, we assessed the level of IL-4 receptor (IL-4R ) and of pSTAT6, a downstream effector of the IL-4R pathway, [50] in control and PYK2 KO Raw264.7 and BMDMs. The pro-tumorigenic phenotype was induced by IL-4 treatment (Experimental Section). As shown in Figure 7B, KO of PYK2 in Raw264.7 cells markedly reduced the level of IL-4R in steady-state conditions or in response to IL-4 treatment, and robustly reduced the phosphorylation of STAT6 in IL-4-treated macrophages. Likewise, the levels of IL-4R and pSTAT6 were significantly reduced in IL-4induced BMDMs from Mϕ-KO compared to Mϕ-WT mice (Figure 7C). These results were further corroborated by IF analysis for IL-4R ( Figure 7D) in WT and PYK2 KO Raw264.7 cells, as well as by IF staining for pSTAT6 ( Figure 7E) and its downstream target CD206 ( Figure 7F). Importantly, significantly lower levels of surface IL-4R (MFI) were also detected by flow cytometry analysis of TAMs from WT tumors in PYK2 KO compared to the WT background ( Figure 7G). Blocking the proteasomal degradation in the Raw264.7 KO cells restored IL-4R and pSTAT6 levels ( Figure 7H), suggesting that PYK2 protects IL-4R from degradation similar to its protective effects on other receptors, including Notch1 ( Figure 4). Collectively, these results suggest that PYK2 regulates IL-4R levels and consequently the IL-4R-pSTAT6 pathway.
Numerous studies have shown that activation of IL-4R induces macrophage polarization into an M2-like phenotype in vitro, and that M2-like macrophages are implicated in protumorigenic effects in vivo. [51] We, therefore, examined the influence of PYK2 ablation on the expression of M2-like markers. WT and PYK2 KO Raw264.7 cells ( Figure 7I) or BMDMs (Figure 7J) were treated with IL-4 and the mRNA expression levels of pSTAT6 target genes including Mrc1, Arg1, Fizz1, Ym1, Ccl22, and Ccl17 were assessed. [52] As shown, ablation of PYK2 reduced the expression levels of all these genes, often reaching significance. To evaluate the effects of PYK2 on the macrophage phenotype in the TME, we used fluorescence-activated cell sorting to isolate TAMs from size-matched breast tumors in WT and PYK2 KO background ( Figure S7A,B, Supporting Information), and analyzed mRNA expression of several pro-tumorigenic polarization markers ( Figure 7K). As shown, PYK2 KO TAMs expressed reduced levels of the following pro-tumorigenic markers: Stab1, Pd-l2 (Pdcd1lg2), Ym1, Irf4 (all p < 0.05) and Il10, Ccl8, Ccl17, Ccl20, Ccl22, Mmp9, and Fizz1 (all p > 0.05). Furthermore, we found a robust decrease of Csf1r mRNA, which plays a crucial role in monocyte and macrophage survival. [53] In addition, we stratified mature F4/80 high macrophages by MHC class II surface expression and found a significant reduction in MHCII low pro-tumorigenic macrophages in PYK2 KO tumors, while the MHCII high (anti-tumorigenic) population was unchanged ( Figure  S7C,D, Supporting Information). Consequently, the MHCII low to MHCII high ratio was significantly reduced upon PYK2 KO (Figure 7L), thus, further suggesting that PYK2 ablation decreases the pro-tumorigenic phenotype.
Together, these results suggest that PYK2 ablation not only inhibited macrophage polarization in vitro, but most importantly also reduced their pro-tumorigenic phenotype in the TME.

PYK2 Ablation Reduces Tumor Angiogenesis
The profound effects of PYK2 ablation on TAM numbers (Figures 1F, 2C,D H,I, and 5F; Figure S5F, Supporting Information) together with the known effects of TAMs on tumor angiogenesis [54] led us to assess the influence of PYK2 ablation either in the BC cells and/or in the TME on angiogenesis using the described different mouse models. CD31 was used as a blood vessel marker to stain tumor sections and evaluate total blood vessel area (Experimental Section). The results were compared between the different tumor models of PYK2 ablation and corresponding WT control. As shown, the blood vessel area was sig-   nificantly reduced when PYK2 was knocked out either in the BC cells (Figure 8A), only in the macrophages ( Figure 8B), or both in the BC and the entire TME ( Figure 8C).
To gain insight into the underlying mechanism of reduced blood vessel area in the PYK2 ablated mouse models, we examined the mRNA levels of key factors known to regulate different stages of angiogenesis/vessel formation, including Vegfa, Il1b, Tymp1, Plau, and Il6, [55,56] by qPCR. Reduced expression levels of these proangiogenic factors were obtained in PYK2-ablated EO771 cells (Figure 8D), Raw264.7 ( Figure 8E), BMDMs (Figure 8F), and TAMs ( Figure 8G). Analysis of tumor samples indicated that ablation of PYK2 either in the BC cells ( Figure 8H), in the TME (Figure 8I), or both in the BC and in the TME (Figure 8I) also reduced the expression of the examined proangiogenic factors. These results suggest that PYK2 can modulate tumor angiogenesis through several mechanisms, and its ablation in the BC cells and/or in the TME can reduce the levels of certain proangiogenic factors and attenuate angiogenesis.
Collectively, our studies uncovered novel mechanisms mediated by PYK2 to control breast cancer-macrophage communication and introduced a unique approach to demonstrate the impact of TAMs on breast cancer progression ( Figure 8J).

Conclusion
The pro-tumorigenic influence of TAMs on breast cancer progression suggests that targeting of TAMs could be a beneficial therapeutic strategy. [15] Indeed, suppression of monocyte recruitment to the TME or inhibition of TAM polarization into protumorigenic macrophages successfully inhibited breast cancer growth in different preclinical models. [4,14,57] In this study we show that depletion of the nonreceptor tyrosine kinase PYK2 markedly reduced the number of macrophages in breast tumors ( Figures 1F, 2C,D,H,I, and 5F; Figure S5F, Supporting Information), and concurrently reduced tumor angiogenesis (Figure 8), and tumor growth (Figures 1E and 2B,G; Figure S2A, Supporting Information; Figure 5E; Figure S5E, Supporting Information).
By using different genetic mouse models and ablation of PYK2 in the BC cells, the entire TME, or selectively in macrophages, we could systematically dissect the discrete influence of PYK2 on BC cells and macrophages alone, and on the BC-macrophage crosstalk signaling. These analyses together with transcriptomic profiling of BMDMs from WT and PYK2 KO mice ( Figure 6D), secretome profiling of PYK2depleted TNBC cells ( Figure 3C; Figure S3A, Supporting Information), and mechanistic studies (Figures 3, 4, 6, and 7), suggest that PYK2 ablation impairs macrophage recruitment and polarization through cell-autonomous mechanisms and by tumormacrophage CCL2/CCR2/CXCR4 and IL-4/IL-4R crosstalk signaling.
Inhibition of tumor growth was observed when PYK2 was ablated either in the BC cells ( Figure 2B; Figure S2A, Supporting Information), in the TME ( Figure 2G), or selectively in macrophages of the TME (Figure 5E), demonstrating the striking influence of TAMs on tumor growth, and the critical role of PYK2 in macrophage-breast cancer communication. KO of PYK2 only in macrophages was sufficient to inhibit tumor growth by ≈35% ( Figure 5E), while KO of PYK2 in BC cells inhibited tumor growth by ≈40%. Nevertheless, KO of PYK2 in both BC and in the TME ( Figure 1E) or both in BC and in macrophages ( Figure S5E, Supporting Information) had no additive influence on tumor growth ( Figure 2K). This unexpected phenotype might be related to the regulatory role of PYK2 in BC-macrophage/monocyte communication, which are usually mediated by receptor-ligand interaction, such as CCL2-CCR2. Hence, inhibition of either ligand secretion or receptor expression could be sufficient to impair receptor downstream signaling, similarly to the effect of ligand and receptor coinhibition.
We previously showed that PYK2 regulates the levels of different cell surface receptors including HER3, cMET, EGFR, AXL, and certain ligands such as IL-8 in breast cancer cells. [16,19,47,58] Other studies demonstrated its effects on secretion of proinflammatory cytokines from macrophages, such as IL-1 and IL-18 [59] or IL-6, TNF-, and IL-12. [60] Interestingly, PYK2 was found to regulate inflammatory response in the gut via direct binding and phosphorylation of IRF5 (Interferon regulating factor 5). [60] Here we show that PYK2 ablation inhibits CCL2 secretion by BC cells (Figure 3D,E) and CCR2 expression in macrophages ( Figure 6E, G, I), thereby impairing CCL2-CCR2 signaling and consequently TAM recruitment. Indeed, previous reports demonstrated the inhibitory effects of CCL2 neutralizing antibodies on breast cancer growth, metastasis, and TAM recruitment. [61] We found that PYK2 regulates Ccl2 transcription in BC cells ( Figure 3F,G,H) through the Notch1 pathway by stabilizing the protein level of N1ICD ( Figure 4A-C; Figure S4A,B, Supporting Information), consistent with previous reports demonstrating the influence of Notch on Ccl2 transcription in breast cancer cells and TAM recruitment in mouse models. [39] Although the underlying mechanism remains to be determined, it could be that PYK2 regulates N1ICD degradation by sequestering the ubiquitin ligase NEDD4-1, which was shown to interact with PYK2 [16] and also to ubiquitinate Notch in Drosophila and skeletal muscle. [62] Importantly, CCL2 can also bind to CCR2-CXCR4 heterodimers. [63] CXCR4 was also reduced in PYK2 KO macrophages ( Figure 6F,H, J), and is known to play a role in cell migration, suggesting that PYK2 regulates TAM recruitment through both cell autonomous-dependent mechanisms and tumor-macrophage communication. Indeed, GSEA analysis of BMDMs RNA-seq data showed significant downregulation of macrophage migration, chemotaxis and integrin signaling ( Figure 6D). The RNA-seq data also highlighted the effect of PYK2 on the IL-4 signaling pathway ( Figure 7A), and further analysis of PYK2 KO macrophages showed a substantial decrease in IL-4R levels and STAT6 phosphorylation in response to IL-4 stimulation (Figure 7B-E,G). STAT6 is a common downstream effector of IL-4R and IL-13R signaling, and its phosphorylation for recruitment of monocytes/macrophages through their CCR2 receptor. In addition to the CCL2/CCR2 axis, macrophage recruitment is regulated by other chemotactic receptors, such as CXCR4, and by cell autonomous migratory properties, which are also affected by PYK2 deficiency. Ablation of PYK2 in macrophages also reduced the level of IL-4R , the main receptor needed for pro-tumorigenic polarization, and robustly reduced STAT6 activation. Consequently, transcriptions of STAT6 target genes are decreased, leading to less pro-tumorigenic TAMs, reduced angiogenesis, and tumor growth. CSL (CBF-1, Suppressor of Hairless, Lag-2), a transcriptional regulator and N1CD binding protein.
is crucial for M2 polarization. [50] Phospho-STAT6 regulates the transcription of M2-characteristic genes including Mrc1, Arg1, Ym1, Mrc1, Ccl17, and Ccl24, [64] and its inhibition attenuates breast cancer growth and enhances immunosuppressive responses of macrophages. [50,65] Indeed, we found significant effects of PYK2 ablation on macrophage polarization in vitro ( Figure 7I,J), on the expression of pro-tumorigenic factors ( Figure 7K) as well as on the MHCII low subpopulation of TAMs ( Figure S7C, Supporting Information), thus demonstrating anti-tumorigenic effects of PYK2 ablation. These results are in agreement with recent reports on the influence of exosomal PYK2 and its binding Rab22a-NeoF1 fusion protein on M2 polarization and lung metastasis of osteosarcoma. [66] Our finding that PYK2 regulates levels of key cell surface receptors including IL-4R, CXCR2, CXCR4, and Notch1 (Figures 4, 6, and 7) and consequently, modulates critical signaling pathways in BC-TAM communication, highlights its therapeutic potential and the clinical implications of our studies. Indeed, CXCR4 blockade significantly reduced chemotaxis of pro-tumorigenic macrophages in oral squamous cell carcinoma [67] and several CXCR4 antagonists, as well as of CCR2 and CSF1R, are currently in clinical trials. [68] Nevertheless, targeting of PYK2 alone might not be sufficient for cancer therapy, whereas combining PYK2 targeting with other therapeutic agents could be beneficial for subsets of breast cancer patients. Previous studies suggest that cotargeting of PYK2 and EGFR could be beneficial for basal-like patients with high EGFR levels, [16] and other studies suggest that immunosuppressive activity of TAMs is associated with upregulation of PD-L1 and immune escape in TNBC. [15] Our finding that PYK2 inhibition reduced the number of TAMs and their pro-tumorigenic phenotype suggests that PYK2 inhibition may sensitize BC to anti-PDL1 blockade. Indeed, previous studies showed that co-inhibition of PYK2 and FAK enhanced the anti-tumor efficacy of an anti-PD-1 immune checkpoint blockade in colorectal tumors, [23] and that PYK2, but not FAK, is required for macrophage recruitment into the TME of PTEN-null glioblastoma multiforme (GBM) to support tumor growth. In this GBM model, PTEN deficiency regulates lysyl oxidase expression, which functions as a macrophage chemoattractant via activation of the integrin 1-PYK2 axis. [69] Hence, development of a highly potent, selective inhibitor for PYK2 could have a therapeutic impact, especially since PYK2 and its most related kinase FAK have overlapping, but still distinct functions, [70,71] particularly in immune cells including macrophages.
TAMs are known for their positive influence on tumor angiogenesis. [49] We found that PYK2 depletion in the cancer cells or macrophages reduced the mRNA expression of several proangiogenic factors (Vegfa, Il1b, Il6, Tymp, and Plau) (Figure 8). Importantly, reduced CCL2 and IL-1 secretion was also reported to influence angiogenesis [55,72] suggesting that PYK2 ablation can affect angiogenesis through different routes.
Overall, we show that PYK2 regulates TAMs in a breast cancer model, and provide evidence that ablation of PYK2 only in macrophages is sufficient to attenuate tumor growth ( Figure 5E), tumor angiogenesis ( Figure 8B), and to reduce TAM numbers ( Figure 5F). These remarkable observations not only highlight the important role of PYK2, but also show, in a unique manner, the critical impact of TAMs on breast cancer progression. Hence, our studies unveil pleiotropic effects of PYK2 on key signaling cascades that modulate macrophage recruitment, polarization, and tumorigenic properties, and highlight its clinical implications in cancer therapy. column was removed from the magnet and placed on top of a 15 mL tube. 5 mL MACS buffer was added and pushed with the stamp through the column. This step was repeated once before the cells were centrifuged at 1400 rpm, 4°C for 7 min and then resuspended in 0.5 mL MACS buffer to be counted.
Mice: PYK2 −/− (PYK2 KO) mice [26] as well as PYK2 f/f mice [25] were established and provided by JA. Girault. Homozygous PYK2 f/f mice were mated with homozygous Cx3cr1 cre mice, [45] generated and provided by S. Jung, to obtain offspring heterozygous for PTK2B and hemizygous for Cx3cr1-Cre. These Cx3cr1-Cre tg/wt /PYK2 f/wt mice were then crossed to generate Cx3cr1-Cre tg/wt /PYK2 f/f , which were finally subjected to experiments (females only). To maintain that line, Cx3cr1Cre tg/wt _PYK2 f/f (Mϕ-KO) were crossed continuously. Mice hemizygous for Cx3cr1-Cre (Cx3cr1-Cre tg/wt ) (Mϕ-WT) served as controls and were obtained by breeding homozygous Cx3cr1 cre mice with wild type C57BL/6 OlaHsd (ENVIGO, Israel). All offspring were genotyped from tail or ear biopsy using the primers listed in Table S3 of the Supporting Information. All mice were in C57BL/6 background. All mice were maintained in a pathogen-free facility, and all animal procedures were approved by the Weizmann Institute's Animal Care and Use Committee (IACUC, investigator accreditation number WIS-060).
Tumor Experiments: Wild-type or Mϕ-WT C57BL/6 mice as well as PYK2 KO mice or Mϕ-KO were orthotopically injected with 0.5 × 10 6 EO771 (WT or PYK2 KO) cells in 50 μL PBS. The cells were implanted in the 3rd mammary fat pad of 8-10-week-old female mice. Tumor size was measured every 3-4 days intervals with a caliper and the volume was calculated using the equation: V = [length × (width) 2 ]/2. When tumors reached the size of ≈0.6-1 cm 3 , animals were euthanized, and tumors were removed. Per experiment, at least three mice per group were injected with tumor cells, experiments were repeated at least twice, and at least six mice were used per group.
For the flow cytometric analyses of tumor composition using sizematched tumors, models associated with PYK2 ablation (EO771 PYK2 KO in WT background or WT EO771 cells in PYK2 KO background) were injected 3 or 4 days, respectively, prior to their corresponding WT control. Tumors were analyzed on day 18 from first injection of the KO models.
RNA Extraction and Real-Time PCR Analysis: Total RNA was extracted using TRI Reagent (Sigma-Aldrich). cDNA was generated from 1 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Subsequently, cDNA was subjected to quantitative real-time PCR using SYBR Green I as a fluorescent dye (Roche) according to the manufacturer's instruction. Real-time PCR anal-ysis was performed on an ABI StepOnePlus 7500 Real-Time PCR system (Applied Biosystems, Invitrogen). All experiments were normalized to Rps29 (mouse) and ACTB (human) RNA levels. Real-time PCR primers were designed using Primer designing tool from NCBI, NIH and were calibrated before use. The primer sequences are listed in Table S4 of the Supporting Information. RNA-Seq and GSEA: RNA was extracted from BMDMs seven days after isolation using TRI Reagent as described above. RNA quality was assessed using Agilent 4200 TapeStation System (Agilent Technologies, Santa Clara, CA). RNA-seq libraries were generated by applying a bulk adaptation of the MARS-seq protocol, as described previously. [77] Libraries were sequenced by the Illumina Novaseq 6000 using SP mode 100 cycles kit (Illumina). Mapping of sequences to the genome and generation of the count matrix was performed by the UTAP pipeline (Weizmann Institute). Libraries normalization, filtration of low count genes and discovery of differentially expressing genes was performed using the edgeR package in R. GSEA was performed using GSEA software (Broad). RNA-seq was performed in three biological replicates. RNA-seq data is available in GEO accession No. GSE193168.
Flow Cytometry of Cells from Tumor Tissue: Eight size-matched tumors per group were chopped and then digested by shaking for 45 min in 5 mL digestion buffer (1 mg mL −1 of collagenase A and 150 μg mL −1 of Hyaluronidase, 1% penicillin-streptomycin in RPMI) at 37°C to obtain single cell suspensions. Tumor fragments were dissociated with a 10 mL pipet several times during the 45 min incubation. Viable cells were counted after filtering the digested samples through 70 μm Falcon cell strainers. Loosely attached cells were collected by washing the strainer with 2 mL PBS. Tumor cells were collected by centrifuging the cell suspension for 5 min at 300 g at 4°C. Cells were washed and suspended in PBS. 5 × 10 6 cells in PBS were subsequently stained in 100 μL FACS buffer (sterile PBS with 3% BSA) against all extracellular target proteins for 30 min on ice, before the cells were washed in FACS buffer, and then fixed. Cells were fixed using the BD Cytofix/Cytoperm Kit according to the manufacturer's instructions. Briefly, the cells were fixed in 50 μL Cytofix for 20 min on ice. The cells were then washed and resuspended in 300-500 μL FACS buffer for analysis, which was carried out on a BD Biosciences FACSAriaIII. Further analyses were performed using FlowJo V10 (TreeStar). For this experiment a live/dead staining was not included.