Immunotherapy against metastatic renal cell carcinoma with mature dendritic cells


Tadaichi Kitamura md phd, Department of Urology, University of Tokyo, 7-3-1, Hongo, Bunkyo, Tokyo 113-8655, Japan. Email:


Objective:  We performed a clinical trial of immunotherapy using autologous mature dendritic cells (DC) pulsed with autologous tumor lysate, for patients with metastatic renal cell carcinoma (RCC).

Methods:  Patients with refractory metastatic RCC were enrolled in the study. All of them received interferon (IFN)-α treatment after nephrectomy and were followed over 3 months prior to this study. Autologous monocyte-derived immature DC were pulsed with lysate from autologous primary tumor as the antigen and keyhole limpet hemocyanin (KLH) as immunomodulator, and cultured in the presence of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin (PG)E2 to generate mature DC. Mature DC were injected intradermally near bilateral inguinal lymph nodes of the patients. A delayed-type hypersensitivity (DTH) test and enzyme-linked immunospot (ELISPOT) assay were performed to evaluate the immunological response. After 4 months from first injection, the clinical effect was evaluated by diagnostic imaging.

Results:  The treatments were well tolerated without significant toxicity by the patients who were an average of 65.7 years old and had multiple metastases in the lung and other organs. One of the two patients developed a positive DTH reaction to tumor lysate and the other patient only to KLH. The patient with a positive DTH reaction to tumor lysate had stable disease in the clinical evaluation.

Conclusions:  We confirmed the safety of DC therapy in this clinical trial. The DTH test revealed that the DC therapy induced immunological response to RCC. On the other hand, it was necessary to reconsider the patient selection criteria.


Metastatic renal cell carcinoma (RCC) remains highly resistant to systemic chemotherapy and radiotherapy.1 Immunotherapy with interferon (IFN)-α and/or interleukin (IL)-2 has been performed, but only a low response rate of 10–20% has been documented.2,3 As the RCC is immunogenic, adoptive immunotherapies, such as those using tumor infiltrating lymphocytes (TIL) and lymphokine activated killer (LAK) cells have been performed before. Nevertheless, these approaches have not shown benefit in comparison with standard cytokine therapies.3–5

Dendritic cells (DC) have a central role in tumor immunity and peptide- or tumor lysate-pulsed DC are able to induce cytotoxic T lymphocytes (CTL) efficiently in vitro by their potent antigen-presenting function. With the aim of amplifying the antitumor effect, the clinical trials of immunotherapy using a large amount of DC generated ex vivo were performed for patients with B-cell lymphoma, prostate cancer, melanoma and other malignant diseases.6–9 Against metastatic RCC, complete response (CR) in three patients and partial response (PR) in five patients out of 174 cases have been reported in 13 papers.10–22 The response rate thus appears to be very low; however, proper evaluation of the effectiveness of the treatments is difficult because the background of the patients and protocols used in those treatments differed from each other. Establishment of an appropriate method for DC therapy against RCC therefore is required for further clinical trials.

Issues that must be considered to design a strategy are: (i) while immature DC were used in some trials, it has been argued that they induce tolerance in CTL rather than activation;23,24 (ii) an autologous tumor antigen was expected to increase the therapeutic effect, and thus, was pulsed to DC before the injection in some trials;8,15,25 and (iii) the migratory efficiency of DC to lymph nodes depends on maturity of DC and routes of injection.26–29 We had already confirmed the safety of the cell-based therapies against chronic myeloid leukemia using blood DC, monocyte-derived immature DC, and monocyte-derived mature DC, which were pulsed with the bcr-abl peptide.30 Only in the case of monocyte-derived mature DC vaccination, all the patients showed a positive result in the delayed-type hypersensitivity (DTH) skin test.31 We also performed a clinical trial for patients with metastatic RCC using monocyte-derived immature DC that were pulsed with autologous tumor lysate. Although we observed some degree of DTH reactivity toward tumor lysate, duration of maintaining positive reactivity was less than 4 weeks.10 Consequently, we considered that the maturation of DC was necessary for effective vaccination against metastatic RCC. In this study, by modifying the previous trial using immature DC against metastatic RCC, we used tumor necrosis factor-α (TNF-α), IL-1β, and prostaglandin (PG)E2 to generate mature DC from monocyte-derived- and then tumor lysate-pulsed-immature DC. The primary endpoint was the evaluation of the safety, and the secondary endpoint was the evaluation of the immunological response to tumor lysate by the DTH test and enzyme-linked immunospot (ELISPOT) assay.31

Materials and methods

Patient selection

The protocol of this clinical trial was approved by the Clinical Institutional Ethics Review Board of the University of Tokyo Graduate School of Medicine in June, 2003 (approval number, 376-[1]). All the patients gave their written informed consent to participate in the study.

Eligibility criteria included histological or radiological documentation of RCC with the primary tumor suitable for resection, age ≥15 years, willingness and ability to undergo surgery, a time interval from prior IFN-α treatment ≥28 days, and Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1. Exclusion criteria consisted of the presence of autoimmune diseases, diabetes mellitus, uncontrolled high blood pressure, active neoplastic diseases other than RCC, active infectious diseases and history of significant cardiac problems.

Preparation of tumor lysate and keyhole limpet hemocyanin

Tumors were placed in normal saline and minced by a cutter after the attached blood clots and necrotic tissues were removed. The minced tissue was suspended in saline and crushed with an ultrasonic disintegrator (Sonifier250, Branson, Danbury, CT, USA). After removal of the undissolved fraction by centrifugation (15 000 g, 10 min), the supernatants were passed through filters (1.2-μm Acrodisc Syringe Filter, Pall Corporation, East Hills, NY, USA), irradiated (120 Gy from 150-Kv X-ray source, using the MBR-1505R2, Hitachi Medical Corporation, Tokyo, Japan) and stored at −80°C. The protein concentration in the tumor lysate was measured by Bradford dye-binding method (Bio-Rad Protein Assay, Bio-Rad, Richmond, CA, USA) using the Absorptiometer (DU530, Beckman Coulter, Fullerton, CA, USA).

Keyhole limpet hemocyanin (KLH; Sigma Chemical, St Louis, MO, USA) was separated from endotoxin by an endotoxin removal filter (ELD96LLC, Pall Corporation). The concentration of endotoxin was confirmed to be less than 0.8 pg/mL by Turbidimetric Time Assay (within the regulation of US Pharmacopeial Convention (USP)).

Culture method of monocyte-derived mature DC

Peripheral blood mononuclear cells (PBMC; 1.1–1.2 × 1010) were collected by 10-L apheresis (AS104, Fresenius AG, Bad Homburg, Germany) and were purified by density gradient centrifugation (Ficoll-Hypaque, Pharmacia, Uppsala, Sweden). The monocyte-rich population was isolated using 50% Percoll density centrifugation (Percoll, Pharmacia). Two-thirds of the isolated population were stored at −80°C in 6% hydroxyethyl starch, 5% dimethyl sulfoxide (CP-1; kindly provided by Kyokuto Pharmacy, Tokyo, Japan) and 4% human serum albumin (kindly provided by Kaketsuken, Kumamoto, Japan) to generate DC used for second and third injections.

Fresh or thawed monocyte-rich cells were incubated with 225 cm2 flasks (Iwaki Glass, Chiba, Japan) for 2 h at 37°C in the growth medium (RPMI-1640 containing 10% autologous serum). Then, nonadherent cells were removed to purify adherent monocytes on the bottom of the flasks. Subsequently, the adherent cells were cultured for 5 days with 1000 U/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF; Leukomax, Novartis, Basel, Switzerland) and 1000 U/mL of IL-4 (CellGenix, Freiburg, Germany). On day 6, a small fraction of cultured cells was confirmed to represent phenotypically immature DC. Then, these cells were pulsed with tumor lysate (100 μg/mL) and KLH (20 μg/mL) for 2 h and cultured for the subsequent 2 days in the presence of TNF-α (CellGenix) at 50 ng/mL, IL-1β at 10 ng/mL (CellGenix), and PGE2 at 1 μg/mL (Sigma). A small fraction of these cells was stained with PE-labeled monoclonal antibodies (mAb) against IgG1 (679.1Mc7, Beckman Coulter), CD14 (M5E2, BD Biosciences, San Jose, CA, USA), CD80 (MAB104, Beckman Coulter), CD83 (HB15a, Beckman Coulter), CD86 (HA5.2B7, Beckman Coulter), human leukocyte antigen (HLA)-DR (B8.12.2 Beckman Coulter), and CCR7 (150503, DAKO, Carpinteria, CA, USA) according to the manufacturer's protocol, and analyzed by flow cytometry using the FACS Calibur (Becton Dickinson, San Jose, CA, USA). For quality control, endotoxin tests and cultures for bacteria and fungi were performed for the end products in our clinical laboratory.

Vaccination schedule

Patients received three vaccinations at 2-week intervals. For injection, tumor lysate-pulsed mature DC were resuspended in 500 μL of normal saline and injected intradermally at the sites near the bilateral inguinal lymph nodes.

Acute and chronic toxicities

Toxicities were assessed by the allergy/immunity section of the National Cancer Institute–Common Toxicity Criteria (NCI-CTC Version 3.0) as follows: (i) acute toxicity, such as fever, transient rash, urticaria, bronchospasm or anaphylactic reaction, was evaluated during the first 1 h after injection; and (ii) chronic toxicity, such as autoimmune reaction, vasculitis or serum disease, was evaluated by physical findings and blood tests (complete blood count, biochemistry, antinuclear and antithyroid receptor antibodies) every 4 weeks. If grade 3–5 toxicities were observed, the clinical trial would be discontinued.

Clinical evaluation

Computed tomography (CT) and bone scintigraphy were performed to evaluate metastases every 4 weeks. At 16 weeks after the first injection, conclusive clinical response was evaluated by the Response Evaluation Criteria in Solid Tumors (RECIST) guideline as follows: (i) CR, the disappearance of all target lesions; (ii) PR, at least a 30% decrease in the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD; (iii) progressive disease (PD), at least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions; and (iv) stable disease (SD), neither sufficient shrinkage to qualify for partial response nor sufficient increase to qualify for progressive disease, taking as reference the smallest sum LD since the treatment started.

DTH test

Tumor lysate-pulsed, KLH-pulsed or unpulsed mature DC (1.5 × 105) were injected into a forearm intradermally every 4 weeks. Forty-eight hours after the injection, the appearance of erythema or induration more than 5 mm in size was considered as positive.


Peripheral blood samples of each patient were obtained before and 4, 8, 12 and 16 weeks after the vaccination. According to the protocol of the Human IFN-γ ELISPOT kit (EL285; R&D Systems, Minneapolis, MN, USA), tumor lysate-pulsed, KLH-pulsed or unpulsed 5 × 104 mature DC and 3 × 105 autologous PBMC obtained each time were added to a 96-well plate precoated with 10 μg/mL of anti-IFN-γ mAb to stimulate IFN-γ-secretion. The plates were incubated for 15 h at 37°C. IFN-γ-secreting cells were detected as a color spot. The number of spots was counted with a stereomicroscope (LEICA MZ8, Leica AG, Heerbrugg, Switzerland).


Patient characteristics

The patients' characteristics are summarized in Table 1. Patient 1 was a 57-year-old man having lung and pancreatic metastases. The IFN-α treatment was terminated 3 months after the initiation, because of the progression of the metastatic tumors. Patient 2 was a 63-year-old man having multiple lung metastases. After the 3-month treatment with IFN-α, he was evaluated to have PD. Patient 3 was a 77-year-old man having multiple lung metastases. His disease had also been evaluated as SD after the 5-month treatment with IFN-α. This treatment, however, was discontinued because of depression.

Table 1.  Patients' characteristics
PatientAgeSexECOGHistologyTNMSites of metastasisPrevious treatments
NephrectomyIFN-α (response)
  1. ECOG, Eastern Cooperative Oncology Group; PD, progressive disease; SD, stable disease; TNM, tumor-node-metastasis.

157Male0Clear/granular cellT3aN0M1Lung, pancreas+3 months (PD)
263Male0Clear cellT3aN0M1Lung+3 months (PD)
377Male0Clear cellT2N0M1Lung+5 months (SD, depression)

Quality of injected DC

Most of the cells cultured for the first 5 days were negative for the monocyte marker (CD14) and moderately positive for the accessory molecules (CD80 and CD86), but negative for CD83 and CCR7, indicating that they differentiated into immature DC (Fig. 1, upper panels). After the culture for a further 2 days as described in Materials and methods, 95% of the cells expressed high levels of CD80, CD83, CD86 and CCR7 (Fig. 1, lower panels). The number of injected DC ranged from 1.0–25 × 106 (Table 2). The tests for bacteria, fungi and endotoxins in cell supernatant were negative in all of the samples.

Figure 1.

Flow cytometric analysis of dendritic cells (DC) before and after co-culture with tumor necrosis factor (TNF)-α, interleukin (IL)-1β and prostaglandin (PG)E2. Histogram plots of DC for first injection before and after co-culture with TNF-α, IL-1β and PGE2 in patient 2. Results were comparable in other patients. All plots are overlayed on the isotype control.

Table 2.  Number of injected dendritic cells (DC), delayed-type hypersensitivity (DTH) reaction and clinical course
 WeeksPatient 1Patient 2Patient 3
  1. In DTH test, erythematous patches in longest × shortest diameter (mm) after 48 h were measured. Sizes of metastases represent sum of the longest diameters (mm) of target lesions (according to Response Evaluation Criteria in Solid Tumors [RECIST] guidelines). Numbers in the parenthesis represent count of metastatic lesions in the organ. KLH, keyhole limpet hemocyanin; ND, not done; TL, tumor lysate.

Number of DC (×106)08725
DTH (kinds of protein) KLH/TLKLH/TLKLH/TL
DTH diameter (mm) 0 × 0/0 × 00 × 0/0 × 00 × 0/0 × 0
Metastatic lesions (no.) Lung (5)/pancreas (1)Lung (5)/bone (1)Lung (4)
Metastases' diameter (cm) 9.6/2.910.6/05.5
Number of DC (×106)2113.3
DTH diameter (mm) 
Metastases' diameter (cm) 
Number of DC (×106)41ND
DTH diameter (mm) 5 × 5/0 × 012 × 12/5 × 6
Metastases' diameter (cm) 10.1/05.3
DTH diameter (mm)85 × 5/0 × 020 × 15/8 × 8
Metastases' diameter (cm) 12/2.65.6
DTH diameter (mm)128 × 5/0 × 025 × 25/10 × 12
Metastases' diameter (cm) 12.2/3.35.5
DTH diameter (mm)167 × 6/0 × 015 × 15/8 × 8
Metastases' diameter (cm) 12.6/4.25.4
Evaluation PDPDSD

Adverse events

There were no abnormalities in blood tests and no grade 3–5 toxicities after injections in all the patients. Transient mild rashes (NCI-CTC: grade 1, less than 10 mm) were observed in the injection sites at every vaccination, except for the first injection in each patient. The rashes disappeared spontaneously within a week.

Clinical course

A conclusive clinical evaluation was determined in CT and bone scintigraphy at 16 weeks after the first injection (Table 2). Although patient 1 dropped out the trial after the first injection, because of the difficulty in visiting our hospital, CT evaluation was performed in another hospital. He had carcinomatous lymphangiosis of the lung and died of respiratory failure 16 weeks after the injection. We evaluated patient 1 as PD. Patient 2 was also evaluated as PD. His lung metastases progressed after 16 weeks and a new metastasis in a lumbar vertebra was observed after 8 weeks. He died of massive pleural effusion and cancerous cachexia 7 months after the first injection. Patient 3 was evaluated as SD. The size of lung metastasis was not changed for 16 weeks after the first injection. Because he had high fever presumably caused by upper respiratory infection at the third injection, we decided to abandon the injection.

Immunological evaluation

Delayed-type hypersensitivity tests were performed in patients 2 and 3 (Table 2). Reaction to KLH in patient 2 was positive from 4–16 weeks. Reaction to KLH and tumor lysate in patient 3 was positive from 4–16 weeks.

In addition, ELISPOT assay was performed in patients 2 and 3 (Fig. 2). KLH- or tumor lysate-reactive cells were not significantly increased in either patients' peripheral blood.

Figure 2.

Enzyme-linked immunospot (ELISPOT) assay. Longitudinal axis represents the number of cells producing IFN-γ from 3 × 105 peripheral blood mononuclear cells (PBMC) co-cultured with 5 × 104 keyhole limpet hemocyanin (KLH)-pulsed, tumor lysate-pulsed or unpulsed mature DC that had been irradiated.


This clinical trial was designed as a modification of our previous trial, in which autologous immature DC were used for patients with metastatic RCC.10 Major changes made in the current trial were as follows: (i) DC were cultured with TNF-α, IL-1β and PGE2 to induce maturation; and (ii) sites of injection were changed from the central parts of the thighs to the inguinal region near the lymph nodes.

The methods to prepare DC to treat metastatic RCC have been variable. In the previous studies in which DC therapies were carried out on 174 cases performed by clinical evaluation, immature DC (unpublished data, Azuma et al, 2002),10–14 mature DC,15–18 allogeneic DC,19,20,22 and DC fused with tumor cells20–22 have been used (Table 3). CR in three and PR in four out of 67 cases with mature DC, and PR in only one out of 43 cases with immature DC, were documented. There were no CR or PR cases in the 64 patients treated with allogeneic DC or DC fused with tumor cells. Although the apparent efficiency rate was low, the use of mature DC seems to be more promising than others. This consideration is consistent with the three properties of mature DC as follows: (i) they are more potent in activating T cells if expressing human leukocyte antigen (HLA) molecules and costimulatory molecules at high levels, such as CD40, CD54, CD80, CD86, HLA-ABC and HLA-DR; (ii) they are more effective in migrating to secondary lymph nodes if expressing CCR7 at a high level; and (iii) they are less likely to induce immunotolerance, which is often the case with immature DC.32,33

Table 3.  List of immunotherapies using DC against renal cell carcinoma (RCC)
YearAuthorNo. of pointsKind of DCAntigenRouteClinical evaluation
  1. Allo, allogeneic; Auto, autogenous; CR, complete response; i.d., intradermal injection; iDC, immature dendritic cell; i.n., intranodal injection; mDC, mature dendritic cell; PR, partial reaction; SD, stable disease (including mixed response and objective response); TC, tumor cell.

2002Azuma10 unpublished data (2002)4Auto iDCAuto TLi.d.0013
2002Oosterwijk-Wakka1112 Auto TLi.d.0084
2003Gitlitz1212 Auto TLi.d.0138
2003Su1310 Tumor RNAi.v., i.d.0010
2004Pandha145 Allo TLi.d.0023
2002Höltl1527Auto mDCAuto TL, Allo TLi.v., i.d.21717
2002Märten1615 Auto TLi.n., s.c.0177
2004Arroyo175 Auto TLi.d.0032
2006Wierecky1820 MUC1 peptide + PADREs.c.12710
Matsumoto (present study)3 Auto TLi.d.0012
2003Märten1912Allo mDCAuto TL, Allo TLi.v., i.d.0048
2004Barbuto2019Allo mDC (fused with)Auto TC, Allo TCi.d00172
2004Avigan2113Auto iDC (fused with)Auto TCs.c.0058
2005Höltl2220Allo mDC (fused with)Auto TCi.n., i.d.00515
Total 177   35169

To date, multiple cytokines, ligands for toll-like receptors and other molecules such as TNF-α, IL-1β, IL-6, PGE2, CD40 ligand, CpG DNA, lipopolysaccharide (LPS) and double-stranded (ds)RNA have been reported to induce maturation of DC.33,34 Before the current clinical trial, we compared the efficiency of various combinations of TNF-α, IL-1β, IL-6, and PGE2 to generate mature DC. Consequently, the DC matured by TNF-α, IL-1β, IL-6 and PGE2 did not express higher levels of CD40, CD80, CD83, CD86 and CCR7 compared to the DC matured by TNF-α, IL-1β and PGE2 without IL-6 (data not shown). The addition of PGE2 to the combination of TNF-α and IL-1β was proven to be most critical for enhancing the maturation of DC. It made DC express the highest level of CCR7 (data not shown). In some reports,35,36 presence of PGE2 was reported to inhibit IL-12p70 production from monocyte-derived DC and to promote the generation of T-helper (Th)2-type responses, which might have a negative impact on antitumor immunity. In others reports, however, monocyte-derived DC matured with PGE2 were reported to enhance Th1-type responses in vitro37 and in vivo.38 In addition, some patients with metastatic RCC had responses in the previous clinical trials using those DC.15,17 Therefore, we selected the combination of TNF-α, IL-1β and PGE2 to generate mature DC in this clinical trial.

As to the routes for injecting DC in the previous reports, i.v., s.c., intradermal, and intranodal injections were chosen for various reasons. Regarding the intranodal injection, there is a risk of injuring the lymph nodes.39 Intradermally-injected DC were reported to have greater migratory capacity than DC injected by other routes.29

As for the sites for DC injection, inguinal, axillary and/or supraclavicular regions near the lymph nodes were chosen in the previous studies.12,15,17 However, information is limited whether the site of injection is really an important determinant to achieve clinical benefit. Moreover, it was difficult to inject DC near regional lymph nodes of various metastatic regions. In this trial, we chose intradermal injection near bilateral inguinal lymph nodes, where we were able to choose the shortest distance to the lymph nodes. With our method, the only side-effects observed were transient rashes. No inguinal lymph node swelling was observed. Nevertheless, the most effective site for DC injection remains to be determined in the future.

In addition, the number of DC is discussed by some investigators to be important for antitumor effect because the low dose of DC can induce the Th2-type response in vitro.40 Similarly, some have reported that increasing the number of DC improved the outcome. However, others have claimed that they could not detect any relation between the immune response and the number of DC.13,16,41–46 The number of injected DC has ranged from 2–100 million in previous studies.33 The mean dose that we injected was 9.2 million (range, 1–25 million), which is almost within the ranges previously used. The optimal dose for DC therapy remains to be further considered.

In the clinical response, disease progression was not controlled in patients 1 and 2. It is generally discussed that the effects of immunotherapy are limited for late-stage disease with a large tumor burden. Therefore, a high response rate might not be expected in this trial. Furthermore, the patients that we selected it turned out (as judged retrospectively) to be so far progressed at registration that we had to discontinue the trial because of their early death.

Immunological evaluations performed in this study included the DTH test and ELISPOT assay. In the previous trials using mature DC against metastatic RCC, Märten16 reported one PR case out of 15 and Arroyo17 reported two cases with mixed response (defined as mixture of regression, progression and persistence of the tumors) out of those five who showed positive DTH reactivity to tumor lysate.

The DTH test could serve as the best immunological monitoring for evaluation of a clinical response, given that longer survival after the DC therapy has been repeatedly linked to positive DTH reactivity.31,47,48 In the DTH tests in this trial, reactions to KLH were positive in SD and PD patients and reaction to tumor lysate was positive only in the SD patient who was still alive as SD for the observation period of 18 months. Of note, positive reaction to tumor lysate continued for 8 weeks in this patient. Although the ELISPOT assay evaluating KLH- or tumor lysate-reactive IFN-γ-secreting cells did not show a positive result in any patients, this assay is less valuable to predict the clinical response than the DTH test.49

Taken together, the long SD status in patient 3 could be caused by the DC therapy, although the possibility remains that the preceding treatment with IFN-α contributed in part because patient 3 was treated for 5 months with IFN-α prior to the registration to this trial. However, he could not continue the therapy because of severe depression. His depression quickly resolved after elimination of IFN-α and the initiation of the DC therapy. In our previous trial using immature DC against metastatic RCC, two of four patients (unpublished data),10 who were evaluated as PD, showed positive DTH reactivity toward tumor lysate, but only transiently. In this trial, we observed continuous positive DTH reaction to tumor lysate in one patient. This contrast suggested that the method used in this trial could be better than that used in our previous trial.

In conclusion, we performed a clinical trial of immunotherapy using autologous mature DC pulsed with tumor lysate for patients with metastatic RCC. Tumor-associated specific immunological reaction for tumor lysate was observed in the SD patient. With no adverse events, tumor regression has not been observed after this treatment. By reconsidering eligibility criteria, this method is expected to prove an effective vaccination.


Department of Urology, Fraternity Memorial Hospital supported the recruitment of patients. Aki Kamijo and Hirokazu Tsuno contributed to technical support of apheresis. Tomomi Nakamura, Yoko Hokama and Mika Matsuhashi are acknowledged for providing excellent technical assistance. Hisamaru Hirai is further acknowledged for obtaining funding and administrative support, and passed away during this study.