The expression of the SPAG9 is associated with various human malignancies. Earlier work revealed a significant association of SPAG9 expression with the early spread of cervical cancer, making it an attractive therapeutic target. Here, the authors investigated the role of SPAG9 in carcinogenesis of squamous cell carcinoma (SCC) of the cervix. Furthermore, they sought to determine whether ablation of SPAG9 expression reduces the tumor growth of cervical SCC in vivo.
A plasmid-based small interfering RNA approach was used to specifically knock down the expression of SPAG9 in SiHa cells derived from SCC of the cervix in vitro and in vivo. Reverse transcriptase polymerase chain reaction, immunofluorescence staining, flow cytometry, cellular growth, colony formation, migration, invasion, and wound healing assays were studied to characterize SPAG9 in vitro. Furthermore, a cervical cancer xenograft model in nude mice was established to investigate whether knockdown of SPAG9 reduces the tumor growth of cervical SCC in vivo.
The results demonstrated that silencing the SPAG9 by small interfering RNA resulted in inhibition of cell growth, colony formation, migration, and invasion. The authors showed for the first time that the knockdown of SPAG9 expression by small interfering RNA significantly suppressed the tumor growth of cervical SCC in vivo.
Cervical cancer is the second most common malignancy and is a leading cause of death among women worldwide.1 In developing countries, nearly 80% of the cases arise because of unavailability of effective screening systems and limited awareness, resources, and medical support.2 The cervical cancer progression from preinvasive to invasive disease is a major challenge to clinical intervention. In this regard, efforts have been made to understand the principles of malignant transformation and tumorigenesis, as this may provide better insight into the process of cancer development and/or tumor suppression.3 Squamous cell carcinoma (SCC) of the cervix is the most frequent malignancy among women and constitutes ∼80% to 90% of total cervical cancer occurrence.1 Thus, the inhibition of invasion and metastasis is of great importance in SCC therapies.
The information available so far indicates that invasion and metastasis is to a large extent attributable to the ability of cells to migrate.4 Over the past decade, there has been progress in the identification of tumor-associated proteins contributing to migratory and invasive properties of tumor cells. In this regard, a unique class of testis proteins, known as cancer testis antigens, are reported to be expressed in a wide range of human tumors.5, 6 It has also been proposed that the aberrant expression of cancer testis antigens in tumors may contribute to various malignant properties, such as immortality, migration, invasion, and metastatic capacity.7 Down-regulation of cancer testis antigen expression in tumor cells might therefore provide an efficient measure to deal with tumor progression.
Recently, we characterized the SPAG9 gene, a new member of the cancer test is antigen family associated with various histotypes of epithelial ovarian cancer (EOC),8 renal cell carcinoma (RCC),9 breast cancer,10 and cervical cancer.11 In addition, gene expression microarray analysis has also reported SPAG9 gene expression in cervical carcinoma cells,12 breast tumors,13 dermatofibrosarcoma protuberans,14 and esophageal adenocarcinoma,15 along with other important and well-studied genes. In addition, our recent study in RCC cells demonstrated that the knockdown of SPAG9 expression with specific small interfering RNA significantly reduced cellular motility and inhibited tumor growth.9
Although a correlation between SPAG9 expression and the early spread of cervical cancer was observed,11 our recent data in EOC,8 RCC,9 and breast cancer10 indicated that SPAG9 may serve as a potential therapeutic target for early stage human tumors. Therefore, in the present study, we used a small interfering RNA approach to selectively knock down the expression of SPAG9 in SiHa tumor cells to confirm the association of SPAG9 with cervical carcinogenesis.
MATERIALS AND METHODS
Cell Cultures and Transient Transfections
Four human cervical cancer cell lines, SiHa (squamous cell carcinoma of cervix), HeLa (adenocarcinoma of cervix), CaSki (epidermoid carcinoma of cervix), and C-33A (carcinoma of cervix) were used in the study. The HeLa cell line was procured from American Type Culture Collection (Manassas, Va) and the CaSki, SiHa, and C-33A cell lines were gifts from Dr. Nicholas Denko (School of Medicine, Stanford University, Palo Alto, Calif). All cancer cells were cultured in recommended medium under standard conditions and were transfected using the transfection reagent Lipofectamine (Invitrogen, Life Technologies, Carlbad, Calif), as described previously.9
RNA Isolation and Reverse Transcriptase Polymerase Chain Reaction Analysis
Total RNA from all cervical cancer cells was prepared using the reagent TRI (Ambion, Inc., Austin, Tex), according to the manufacturer's protocol and amplified with SPAG9-specific primers, as described earlier.9 The polymerase chain reaction (PCR) products were electrophoresed on 1% agarose gels containing ethidium bromide and photographed under ultraviolet light. β-Actin was used as an internal control in each reaction. The PCR products were cloned into TOPO vector (Invitrogen) to confirm the DNA sequence.
Immunofluorescence and Flow Cytometric Analysis
For immunofluorescence assay, cervical cancer cells were harvested and analyzed for SPAG9 localization using anti-SPAG9 antibody or control immunoglobulin (Ig) G, as described earlier.8 Briefly, fixed and permeabilized or live cancer cells were incubated with anti-SPAG9 antibody, followed by antirat IgG fluorescein isothiocyanate conjugate as secondary antibody (Jackson ImmunoResearch, West Grove, Pa). The slides were subsequently washed, mounted in glycerol/phosphate-buffered saline (PBS) (9:1) and observed under a Nikon Eclipse E 400 microscope (Nikon, Fukok, Japan). Flow cytometric analysis was carried out on cancer cells using anti-SPAG9 antibody, as previously described.8 Data acquisition and analysis were done using WinMDI (version 2.8; Purdue University, West Lafayette, Ind) software. Side scatter versus forward scatter gate analysis was done in all cell lines. Cells stained with secondary antibody only were used to account for the background fluorescence.
Analysis of RNA Interference Activity
The BS/U6 vector was used to construct SPAG9 small interfering RNA (SPAG9 small interfering RNA and SPAG9 small interfering RNA-I) and control small interfering RNA (scrambled SPAG9), as described previously.16 SiHa cells, SCC-derived cervical cancer cells, were used to determine small interfering RNA-mediated knockdown of SPAG9. Briefly, cells were seeded at a density of ∼5 × 105 in a 35-mm dish plate and cultured for 16 hours. At 60% confluency, cells were transfected with 6 μg of SPAG9 small interfering RNA, SPAG9 small interfering RNA-I, or control small interfering RNA plasmids for 3 hours, and subsequently the culture medium was replaced with Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cell lysate of transfected cells was prepared, resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Hybond-P, Amersham Biosciences, Cowley, UK), and probed using anti-SPAG9 antibody, as described earlier.9
Cell Growth and Colony Formation Assays
Cell growth and colony formation assays were done, as described previously.9 Briefly, SiHa cells, transfected with SPAG9 small interfering RNA or control small interfering RNA, were trypsinized, counted, and seeded at a density of ∼5 × 105 cells in 35-mm dish plates. Cell growth was determined by counting the number of cells at 24 hours, 48 hours, and 72 hours using a hemocytometer. For colony formation assay, SiHa cells were grown and treated with SPAG9 small interfering RNA or control small interfering RNA and plated at low densities in 35-mm dishes (∼4 × 102 to 103 cells/plate) in triplicates. Cells were allowed to grow for 9 to 12 days, and the colonies were fixed with 5% glutaraldehyde in PBS, stained with 5% crystal violet, and counted manually.
Migration and Invasion Assays
The ability of the SiHa cells transfected with 6 μg of SPAG9 small interfering RNA or control small interfering RNA to migrate through a filter or to invade through a biological barrier was determined by transwell insert chambers (Becton Dickinson, Franklin Lakes, NJ) with 8-μm pore filters. SiHa cells at a density of 2 × 105 cells/mL were seeded in the upper chambers with 200 μL serum-free DMEM, and the lower wells were filled with 750 μL DMEM, with 10% FBS as an inducer for cell migration. For the invasion assay, a matrix barrier was formed by coating Matrigel at 5 mg/mL in serum-free DMEM. After an incubation period of 48 hours at 37°C, the cells that migrated to the lower surface of the filter were fixed with 5% glutaraldehyde in PBS for 10 minutes, stained with 0.5% toluidine blue (Sigma-Aldrich, St. Louis, Mo), and counted. Each experiment was carried out in triplicate in 3 independent experiments to ensure consistency. The statistical significance of the results was determined using Student t test. A P value of <.05 was considered statistically significant.
Monolayer Wound Healing Assay
Wound healing assay was done to determine the changes in cell motility, as described previously.9 Briefly, SiHa cells transfected with 6 μg of SPAG9 small interfering RNA or control small interfering RNA were seeded at a density of 2 × 106 in 35-mm dish plates. At 100% confluency, cells were scratched with a 200-μL filter tip to create an artificial wound and photographed at 0 hours and at a time interval of 12 hours, 24 hours, and 48 hours. For validation of data, every experiment was done in triplicates and repeated 4 times.
In Vivo Effects of SPAG9 Small Interfering RNA on the Growth of Cervical Xenograft in Nude Mice
For in vivo studies, human tumor xenografts were established in 4-week-old athymic nude mice (Nii:NIH [S] [nu/nu]). Two groups of 6 mice were injected with 1 × 107 SiHa cells subcutaneously to generate a primary tumor. Administration of small interfering RNA plasmid was initiated when the tumor volume was ∼50-100 mm3. Fifty micrograms of plasmids in 200 μL of PBS were injected into the tail vein of the mice followed by a booster dose of 25 μg of plasmid, twice a week for 6 weeks. Tumors were monitored for a total of 42 days. Tumor volume was calculated by using the formula V = π 6 × Dl × Ds2, where V is volume, Dl is the largest diameter, and Ds is the smallest diameter. Mice were sacrificed after 42 days, and tumors were excised, weighed, and processed for immunohistochemical analysis of SPAG9 and proliferating cell nuclear antigen (PCNA) expression.
Tumor tissues excised from the control small interfering RNA and SPAG9 small interfering RNA-injected nude mice were fixed in formalin. Tissue sections (3-4 μm) were treated with 1% hydrogen peroxide to block the endogenous peroxidase activity and subsequently blocked with 5% normal goat serum. Tissue sections were then incubated with anti-SPAG9 antibody at 4°C overnight, followed by washing with PBS and incubation with horseradish peroxidase-conjugated goat antirat IgG (Jackson ImmunoResearch) for 2 hours at room temperature. Immunoreactivity was observed using chromogen 0.05% 3,3′-diaminobenzidine (Sigma Aldrich, St. Louis, Mo). Six random fields of each tissue section were examined by counting >500 cells under ×400 magnification.
The significance of the in vitro and in vivo data was determined by Student 2-tailed t test using the SPSS 16.0 statistical software package (SPSS Inc., Chicago, Ill). P < .05 was considered statistically significant. All experimental data are presented as mean ± standard error.
Expression of SPAG9 mRNA in Cervix Cancer Cell Lines
We previously reported that SPAG9 gene is expressed in most patients with SCC of the cervix.11 In the present study, we investigated the expression of the SPAG9 gene in various cervical cancer cell lines: SiHa (SCC of cervix), HeLa (adenocarcinoma of cervix), CaSki (epidermoid carcinoma of cervix), and C-33A (carcinoma of cervix). The total RNA was isolated and cDNA was synthesized from SiHa, HeLa, CaSki, and C-33A cells. After cDNA synthesis, to avoid any false-positive results attributable to the amplification of contaminated genomic DNA in the cDNA preparation, all SPAG9 primers were designed to amplify exons separated by an intron. SPAG9 mRNA was detected in all 4 cell lines and testis by reverse transcriptase (RT) PCR, as illustrated in Figure 1A. The PCR amplicon in cancer cells was the same as in testis. The PCR products from these cancer cells were cloned in TOPO vector and confirmed by nucleotide sequencing. The DNA sequence analysis of the PCR product showed no mutation. cDNA quality was checked by using β-actin amplification as an internal control (Fig. 1A). Hence, these results suggest that SPAG9 gene expression was independent of histologic cell types of cervical cancer.
SPAG9 Protein Expression in Cervical Cancer Cell Lines
Earlier, we reported that SPAG9 protein expression was associated with a significant proportion of patients with SCC of the cervix.11 Therefore, in the present study, we investigated the SPAG9 protein expression in various histologic types of cervical cancer cells by indirect immunofluorescence and flow cytometric analysis using anti-SPAG9 antibodies. As revealed in representative photomicrograph in Figure 1B, strong SPAG9 immunoreactivity was observed in cervical cancer cells. The results showed that in fixed and permeabilized cervical cancer cells, SiHa (Fig. 1B[a]), HeLa (Fig. 1B[c]), CaSki (Fig. 1B[e]), and C-33A (Fig. 1B[g]), SPAG9 protein was abundant in the cytoplasm; however, distinct surface localization was observed in live cervical cancer cells: SiHa (Fig. 1B[b]), HeLa (Fig. 1B[d]), CaSki (Fig. 1B[f]), and C-33A (Fig. 1B[h]). Subsequently, these results were confirmed by flow cytometric analysis (Fig. 2). Side scatter versus forward scatter gate analysis was carried out for all the experiments to avoid debris (data not shown). In the control cells, no or very low surface distribution 0.59% (Fig. 2A), 0.37% (Fig. 2C), 0.46% (Fig. 2E), and 0.27% (Fig. 2G) fluorescence intensity was observed, whereas 93% (Fig. 2B), 94% (Fig. 2D), 95% (Fig. 2F), and 97% (Fig. 2H) fluorescence intensity or displacement of fluorescence on the x axis was observed in SiHa, HeLa, CaSki, and C-33A cells, respectively, distinctly showing surface localization of SPAG9 protein. Hence, these results showed that each of these histologic types of cervix cancer cells exhibit SPAG9 protein expression, which may participate in cancer growth and early spread, as reported earlier in cancer patients with SCC of the cervix.11
Reduction of SPAG9 Expression in SiHa Cells by RNA Interference
A small interfering RNA approach was used to selectively knock down the expression of SPAG9 in SCC-derived SiHa cervical cancer cells in vitro and in vivo. We designed and synthesized the 2 complementary oligonucleotides encoding short hairpin transcripts directed against a portion of the SPAG9 mRNA (GenBank Accession No. X91879) and cloned as described earlier.16 To investigate the potential of SPAG9 as a therapeutic target for cervical carcinoma, our approach was to use SPAG9 small interfering RNA to inhibit SPAG9 expression and study its effect on cell growth and in cellular behaviors in cancer cells. As illustrated in Figure 3A, 2 SPAG9-specific small interfering RNA targets (SPAG9 small interfering RNA-I and SPAG9 small interfering RNA) reported earlier9 were analyzed. Western blotting analysis revealed that SPAG9 small interfering RNA significantly resulted in ablation of SPAG9 protein expression in comparison to SPAG9 small interfering RNA-I; however, there was some residual SPAG9 protein expression detected. Furthermore, densitometric analysis revealed 70% reduction in SPAG9 protein expression in SPAG9 small interfering RNA-treated cells as compared with SPAG9 small interfering RNA-I and control small interfering RNA-treated cells (Fig. 3B). Therefore, subsequently all in vitro and in vivo assays were carried out using SPAG9 small interfering RNA. SiHa cells were transfected with SPAG9 small interfering RNA, which resulted in a gradual and significant decrease in cell growth (Fig. 3C). After 72 hours, cell growth was reduced to 29%. Similarly, the colony formation was significantly (P < .0001) inhibited by treatment with SPAG9 small interfering RNA, but not with control small interfering RNA (Fig. 3D). The number of cell colonies treated with SPAG9 small interfering RNA was significantly reduced for various cell numbers seeded for SiHa (range of 28%-32% for 400-1000 cells). These results indicate that vector-based RNA interference could effectively suppress SPAG9 expression that apparently reduces cell growth and colony-forming ability in cervical cancer cells.
Depletion of SPAG9 Suppresses Metastatic Properties of Cervical Cancer Cells In Vitro
Tumor metastasis comprises multiple steps, wherein tumor cells need to express a variety of properties, including increased motility and invasive capacity, to complete the metastatic process. Because the cellular motility is considered an important step in the invasive processes of metastasis, the effect of SPAG9 small interfering RNA on cell migration was examined. Transwell migration assays were carried out using 8-μm inserts, which revealed 68% inhibition in the motility potential of SiHa cells transfected with SPAG9 small interfering RNA (Fig. 4A). The histogram representation also reveals a significantly lower number of cells having migrated through inserts (P < .0001).
Subsequently, we evaluated the ability of SiHa cells transfected with control small interfering RNA or SPAG9 small interfering RNA to invade through an artificial extracellular matrix in a Matrigel invasion assay. Similar to the migration assay, SPAG9 small interfering RNA inhibited the invasion of SiHa cells by 69% (Fig. 4B). As shown in Figure 4B, the histogram representation shows that a significantly lower number of SiHa cells invaded through a reconstituted basement membrane barrier (Matrigel), indicating that the invasive potential of SiHa cells was severely affected in SPAG9 small interfering RNA-transfected cells (P < .0001).
Furthermore, to investigate the antimetastatic activity of SPAG9 small interfering RNA, we assessed the effect of SPAG9 small interfering RNA on the migration of SiHa cells in the wound healing assay. The migration of SiHa cells was compared between control small interfering RNA and SPAG9 small interfering RNA transfected cells after creating scratch wounds at 12 hours, 24 hours, and 48 hours. As shown in Figure 4C, the closing of the scratch wound in SPAG9 small interfering RNA transfected cells was not complete even after 48 hours, whereas cells transfected with control small interfering RNA successfully closed the entire wound in 24 hours. These results collectively indicate that the SPAG9 small interfering RNA effectively reduces the migration ability of SiHa cells.
RNA Interference-Mediated Inhibition of SPAG9 Decreases Human Cervical Tumor Growth in Nude Mice
Our findings that reduction of SPAG9 expression by RNAi significantly inhibited colony formation led us to evaluate its effect on tumor growth in nude mice in vivo. The dose and route of SPAG9 small interfering RNA administration was determined from our previous study documenting in vivo efficacy of SPAG9 small interfering RNA against renal cancer cells.9 The effect of SPAG9 small interfering RNA or control small interfering RNA on SiHa cervical tumor in nude mice was continuously monitored for 42 days. In Figure 5A, a representative photograph shows reduced tumor growth in SPAG9 small interfering RNA-treated mice compared with control small interfering RNA-treated mice. Figure 5B shows the average tumor volume over the total 42 days of study between these 2 groups. Mice injected with SPAG9 small interfering RNA had sustained a significant tumor growth arrest compared with mice administered control small interfering RNA, as shown in Figure 5C. On average, SPAG9 small interfering RNA treatment resulted in decreased tumor growth by 68% at Day 24 (P = .03), 73% at Day 36 (P = .005), and 78% at Day 42 (P < .0001). Consistent with tumor volume data, the average wet weight of the tumor was significantly (P < .0001) reduced in SPAG9 small interfering RNA-treated mice compared with mice administered control small interfering RNA (Fig. 5D).
Furthermore, to investigate whether the inhibition of tumor growth is associated with the down-regulation of SPAG9 expression, the xenograft tumors were dissected and processed for immunohistochemical staining of SPAG9 protein expression. As shown in Figure 6, the SPAG9 protein was down-regulated in SPAG9 small interfering RNA-administered xenograft (Fig. 6D), compared with high SPAG9 expression in the control small interfering RNA-administered tumors (Fig. 6C). In addition, to test whether SPAG9 small interfering RNA-mediated inhibition of SiHa cancer growth in vivo was associated with reduced cell proliferation, tumor tissues were processed and probed for PCNA expression. As shown in Figure 6, a significant reduction of PCNA expression (63%; P < .0001) was observed in tumors from SPAG9 small interfering RNA-treated mice (Fig. 6B) compared with mice treated with control small interfering RNA (Fig. 6A). These results further strengthen the significant effect of SPAG9 suppression on SiHa cancer growth and suggest that SPAG9 may be a molecular target for cancer treatments.
Cervical cancer remains a major gynecologic malignancy, causing a rise in the incidences of cancer death among women because of metastasis.1 Especially in developing countries, most cervical cancer cases arise because of unavailability of screening methods and proper medical infrastructure, which puts the patients at higher risk of invasiveness of the disease.2 Invasive neoplasms arise from transformed cells through clonal expansion of cells having a selective advantage.17 Eventually genomic instability, both genetic and epigenetic, enhances the process of cellular proliferation and invasive potential features of cancer cells. Cervical cancer is a heterogeneous group of neoplasms that includes different histologic subgroups, each with its own underlying molecular genetic events.
Small interfering RNA is a novel approach to induce gene silencing through induction of RNA interference. This specific method has emerged as 1 of the most promising strategies for therapeutic product development. Its wider application with high efficiency18 and specificity19, 20 could have high impact and clinical relevance to any disease by manipulating gene expression.21 The present study for the first time reports the effective use of vector-based small interfering RNA-mediated knockdown of SPAG9 expression to address the possible role of SPAG9 in metastasis. Our findings suggest that the small interfering RNA-directed transcriptional silencing is a promising tool for suppression of SPAG9 gene function and illustrate the potential of its application in cancer treatment.
Recently, we reported the SPAG9 gene, which is involved in the c-Jun N terminal kinase (JNK) signaling module22, 23 and functions as a scaffolding protein. SPAG9 specifically binds to JNK24 that plays an important regulatory role in several physiologic processes, including cell survival, proliferation, apoptosis, and tumor development.25, 26 Recent studies have indeed indicated the role of SPAG9 in the early spread of renal cell carcinoma9 and breast cancer.10 Furthermore, SPAG9 was found to be associated with malignancy in various histotypes of epithelial ovarian cancer.8 In addition, we also reported an association of SPAG9 expression with early stages of cervical cancer in clinical specimens.11 These observations prompted us to investigate the role of SPAG9 expression in the tumorigenesis of cervical cancer. The metastasis is associated with increased migratory and invasive properties; therefore, we investigated the role of SPAG9 expression in cellular motility and invasiveness in SiHa cells derived from SCC of the cervix. The important finding of this study is that SPAG9 expression in cervical cancer is associated with cellular growth, cell migration, and invasion.
To date, the expression of several cancer testis antigen genes such as MAGE, GAGE-3/6, LAGE, and PRAME has been reported in the HeLa cervical cancer-derived cell line.25 In contrast, no expression of BAGE, GAGE-1/2, and NY-ESO-1 genes was observed in HeLa cells. Expression of CAGE-1 and CRT2 has also been reported in cervical cancer cell lines.26 However, association of cancer testis antigens with the malignant properties of SCC of the cervix has not been established so far. Because SCC of the cervix is a leading cause of death in women worldwide,1 in this current study we used SiHa cells derived from SCC of the cervix to investigate the functional role of SPAG9 in tumorigenesis of cervical cancer. SPAG9 gene expression was detected in different histotypes of cervical cancer cell lines by RT-PCR. Furthermore, sequence analysis of the RT-PCR products from different cervical cell lines revealed a homologous nucleotide sequence as SPAG9 cDNA and did not show any mutation within SPAG9 cDNA. To assess whether SPAG9 plays a critical role in the malignancy of cervical cancer cells, we knocked down the expression of endogenous SPAG9 in SiHa cells. SPAG9 small interfering RNA successfully suppressed SPAG9 expression in cervical cancer cells, leading to a suppression of cellular growth, migration, and invasion of SiHa cervical cells in vitro. Our findings revealed that knockdown of SPAG9 expression by small interfering RNA significantly suppressed cellular motility and tumor growth of cervical SCC in vivo. In this regard, a recent study also revealed that knockdown of SMYD3 expression by small interfering RNA significantly reduced cell proliferation, migration, and invasion of HeLa cells.27 We further confirmed that SPAG9 small interfering RNA suppressed tumor growth in a xenograft cervical tumor model in immune-compromised mice. Our study demonstrated that small interfering RNA directed against SPAG9 successfully inhibited the metastatic potential of SiHa cervix cells.
In summary, the present study provides evidence indicating a critical and specific role of SPAG9 in malignant properties of cervical carcinoma. Furthermore, our data demonstrate that plasmid-mediated RNA interference of SPAG9 successfully inhibited the expression of SPAG9 in in vitro and in vivo models of cervical cancer, leading to several antitumor activities such as inhibitory effects on cell proliferation, migration, invasion, and tumor growth. We successfully established that the suppression of SPAG9 expression can effectively decrease the cellular motility and tumor growth of cervical SCC in vivo. These findings suggest that the RNA interference approach can be an effective therapeutic strategy for cervical cancer and therefore warrant future investigations in human clinical trials. The results support the potential role of SPAG9 in tumorigenesis of cervical cancer cells, which confirmed the association of SPAG9 expression in cervical cancer patients.11
Conflict of Interest Disclosures
Supported by the Cancer Research Program, Associated Cancer Center for Immunotherapy, Department of Biotechnology, Government of India.