Supplementary Figure 3 - Expression of VEGFR-1 and IL-1ß in TAMs in PAN02 tumors.

Effects of VEGFR-1 signaling ablation on glucose/insulin tolerance and insulin production by the pancreas during obesity.

Supplementary Figure 5 - Effects of VEGFR-1 signaling ablation on PAN02 tumor progression in body-weight matched mice.

Supplementary Table 1: Protein quantification of cytokines in tumors and plasma of WT and Flt1TK-/- obese using ELISA; Supplementary Table 2: Quantification of the expression of IL-1ÃŽÃ,² and VEGFR-1 in TAMs from PAN02 tumors by mmunofluorescence.

Supplementary Figure 1 - A) Vessel density in PAN02 tumors from lean and obese mice, and effect of VEGFR-1-TK-deletion on tumor vessel density and hypoxia markers in obese mice. Supplementary Figure 2 - Gene expression of M1 / M2 markers in TAMs isolated from PAN02 tumors in lean and obese mice.

Supplementary Tables S1 - S6. Supplementary Table S1. CT values for demosplasia-related genes in PAN02 tumors. Data obtained from PCR array. Supplementary Table S2. CT values for demosplasia-related genes in AK4.4 tumors. Data obtained from PCR array. Supplementary Table S3. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S4. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S5. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25. Supplementary Table S6. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25.

Supplementary Figure 7 - Effects of VEGFR-1 signaling ablation on IGF-1R/IR signaling pathways in PAN02 and E0771 tumors implanted in obese mice; Supplementary Figure 8 - Additional effects of VEGFR-1 signaling ablation and metformin on tumor metabolism and vessel density in obese mice.

Supplementary Figure 10 - Additional measurements of PlGF and VEGF-B; Supplementary Figure 11 - Additional effects of PlGF deletion on obesity-induced tumor progression and systemic metabolism.

Effects of VEGFR-1 signaling ablation on E0771 tumor growth, vascular and immune environment in lean and obese mice.

Effects of VEGFR-1 signaling ablation on body weight gain, immune cell infiltration and vasculature in adipose tissues during obesity.

Abstract Antigen presentation plays a major role in tumor cell recognition and targeting by immune cells, and is critical to the success of many cancer immunotherapies. How the abnormal tumor microenvironment affects tumor cell antigen presentation is unclear. Hypoxia is a prevalent feature of the tumor microenvironment. Here, we showed that the expression of major histocompatibility complex class I (MHCI) is associated with regions of hypoxia in human breast tumors. The association between hypoxia and MHCI is independent of the breast tumor hormone receptor and HER2 expression status. In vitro studies revealed that hypoxia directly regulates the expression levels of MHCI along with other components of the antigen presentation machinery. Multiple kinase regulators of MHCI expression are responsive to hypoxia. These results suggest that hypoxia effects on cancer cell antigen presentation may be a potential mechanism of tumor immune evasion and treatment resistance. Citation Format: Mei Rosa Ng, Francesco Sabbatino, Mark Duquette, Kamila Naxerova, Mark Badeaux, Gino B. Ferraro, Shan M. Chin, Divya Bezwada, Elena F. Brachtel, Soldano Ferrone, Rakesh K. Jain. Hypoxia regulation of antigen presentation machinery expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-057.

Anti-vascular endothelial growth factor (VEGF) therapy has failed to improve survival in patients with breast cancer (BC). Potential mechanisms of resistance to anti-VEGF therapy include the up-regulation of alternative angiogenic and proinflammatory factors. Obesity is associated with hypoxic adipose tissues, including those in the breast, resulting in increased production of some of the aforementioned factors. Hence, we hypothesized that obesity could contribute to anti-VEGF therapy's lack of efficacy. We found that BC patients with obesity harbored increased systemic concentrations of interleukin-6 (IL-6) and/or fibroblast growth factor 2 (FGF-2), and their tumor vasculature was less sensitive to anti-VEGF treatment. Mouse models revealed that obesity impairs the effects of anti-VEGF on angiogenesis, tumor growth, and metastasis. In one murine BC model, obesity was associated with increased IL-6 production from adipocytes and myeloid cells within tumors. IL-6 blockade abrogated the obesity-induced resistance to anti-VEGF therapy in primary and metastatic sites by directly affecting tumor cell proliferation, normalizing tumor vasculature, alleviating hypoxia, and reducing immunosuppression. Similarly, in a second mouse model, where obesity was associated with increased FGF-2, normalization of FGF-2 expression by metformin or specific FGF receptor inhibition decreased vessel density and restored tumor sensitivity to anti-VEGF therapy in obese mice. Collectively, our data indicate that obesity fuels BC resistance to anti-VEGF therapy via the production of inflammatory and angiogenic factors.

Abstract It remains unclear how obesity worsens treatment outcomes in patients with pancreatic ductal adenocarcinoma (PDAC). In normal pancreas, obesity promotes inflammation and fibrosis. We found in mouse models of PDAC that obesity also promotes desmoplasia associated with accelerated tumor growth and impaired delivery/efficacy of chemotherapeutics through reduced perfusion. Genetic and pharmacologic inhibition of angiotensin-II type-1 receptor reverses obesity-augmented desmoplasia and tumor growth and improves response to chemotherapy. Augmented activation of pancreatic stellate cells (PSC) in obesity is induced by tumor-associated neutrophils (TAN) recruited by adipocyte-secreted IL1β. PSCs further secrete IL1β, and inactivation of PSCs reduces IL1β expression and TAN recruitment. Furthermore, depletion of TANs, IL1β inhibition, or inactivation of PSCs prevents obesity-accelerated tumor growth. In patients with pancreatic cancer, we confirmed that obesity is associated with increased desmoplasia and reduced response to chemotherapy. We conclude that cross-talk between adipocytes, TANs, and PSCs exacerbates desmoplasia and promotes tumor progression in obesity. Significance: Considering the current obesity pandemic, unraveling the mechanisms underlying obesity-induced cancer progression is an urgent need. We found that the aggravation of desmoplasia is a key mechanism of obesity-promoted PDAC progression. Importantly, we discovered that clinically available antifibrotic/inflammatory agents can improve the treatment response of PDAC in obese hosts. Cancer Discov; 6(8); 852–69. ©2016 AACR. See related commentary by Bronte and Tortora, p. 821. This article is highlighted in the In This Issue feature, p. 803

Abstract Purpose: Obesity promotes pancreatic and breast cancer progression via mechanisms that are poorly understood. Although obesity is associated with increased systemic levels of placental growth factor (PlGF), the role of PlGF in obesity-induced tumor progression is not known. PlGF and its receptor VEGFR-1 have been shown to modulate tumor angiogenesis and promote tumor-associated macrophage (TAM) recruitment and activity. Here, we hypothesized that increased activity of PlGF/VEGFR-1 signaling mediates obesity-induced tumor progression by augmenting tumor angiogenesis and TAM recruitment/activity. Experimental Design: We established diet-induced obese mouse models of wild-type C57BL/6, VEGFR-1 tyrosine kinase (TK)-null, or PlGF-null mice, and evaluated the role of PlGF/VEGFR-1 signaling in pancreatic and breast cancer mouse models and in human samples. Results: We found that obesity increased TAM infiltration, tumor growth, and metastasis in pancreatic cancers, without affecting vessel density. Ablation of VEGFR-1 signaling prevented obesity-induced tumor progression and shifted the tumor immune environment toward an antitumor phenotype. Similar findings were observed in a breast cancer model. Obesity was associated with increased systemic PlGF, but not VEGF-A or VEGF-B, in pancreatic and breast cancer patients and in various mouse models of these cancers. Ablation of PlGF phenocopied the effects of VEGFR-1-TK deletion on tumors in obese mice. PlGF/VEGFR-1-TK deletion prevented weight gain in mice fed a high-fat diet, but exacerbated hyperinsulinemia. Addition of metformin not only normalized insulin levels but also enhanced antitumor immunity. Conclusions: Targeting PlGF/VEGFR-1 signaling reprograms the tumor immune microenvironment and inhibits obesity-induced acceleration of tumor progression. Clin Cancer Res; 22(12); 2993–3004. ©2016 AACR.

BACKGROUND:Pancreatic ductal adenocarcinoma (PDAC) is a highly desmoplastic tumor with a dismal prognosis for most patients. Fibrosis and inflammation are hallmarks of tumor desmoplasia. We have previously demonstrated that preventing the activation of pancreatic stellate cells (PSCs) and alleviating desmoplasia are beneficial strategies in treating PDAC. Metformin is a widely used glucose-lowering drug. It is also frequently prescribed to diabetic pancreatic cancer patients and has been shown to associate with a better outcome. However, the underlying mechanisms of this benefit remain unclear. Metformin has been found to modulate the activity of stellate cells in other disease settings. In this study, we examine the effect of metformin on PSC activity, fibrosis and inflammation in PDACs.METHODS/RESULTS:In overweight, diabetic PDAC patients and pre-clinical mouse models, treatment with metformin reduced levels of tumor extracellular matrix (ECM) components, in particular hyaluronan (HA). In vitro, we found that metformin reduced TGF-ß signaling and the production of HA and collagen-I in cultured PSCs. Furthermore, we found that metformin alleviates tumor inflammation by reducing the expression of inflammatory cytokines including IL-1β as well as infiltration and M2 polarization of tumor-associated macrophages (TAMs) in vitro and in vivo. These effects on macrophages in vitro appear to be associated with a modulation of the AMPK/STAT3 pathway by metformin. Finally, we found in our preclinical models that the alleviation of desmoplasia by metformin was associated with a reduction in ECM remodeling, epithelial-to-mesenchymal transition (EMT) and ultimately systemic metastasis.CONCLUSION:Metformin alleviates the fibro-inflammatory microenvironment in obese/diabetic individuals with pancreatic cancer by reprogramming PSCs and TAMs, which correlates with reduced disease progression. Metformin should be tested/explored as part of the treatment strategy in overweight diabetic PDAC patients.

Abstract Background: Obesity associates with angiogenesis and increased macrophage infiltration in adipose tissues during weight gain. Whether these effects also occur in cancer to promote tumor progression in obese condition remains unclear. We have shown that the vascular endothelial growth factor receptor-1 (VEGFR-1) pathway can modulate tumor angiogenesis and recruitment of tumor-associated macrophages (TAMs). Here, we tested the emerging hypothesis that obesity enhances tumor progression and metastasis by augmenting angiogenesis and TAM recruitment via activation of the VEGFR-1 pathway. Methods: We used a high-fat diet-induced obesity model in either wild type (WT) or VEGFR-1 tyrosine kinase null (Flt1TK-/-) C57BL/6 mice. Then, we implanted orthotopically syngeneic pancreatic (PAN02) or breast (E0771) carcinomas. We evaluated the role of VEGFR-1 activity on systemic metabolism, tumor angiogenesis and immune environment, and tumor growth and metastasis. Results: Obesity increased p38-MAPK activation and TAM infiltration, tumor growth (p = 0.001) and metastasis (p = 0.035) in PAN02 tumors. VEGFR-1 inhibition reduced tumor growth (p = 0.007) and metastasis (p = 0.017) in obese but not lean mice. This was associated with a decreased p38-MAPK activity and a shift in TAM polarization towards the M1 phenotype with reduced secretion of pro-tumor cytokines, but no change in vascular density or number of TAMs. In the E0771 model, VEGFR-1 inhibition reduced MMP-9 expression and decreased lung metastatic burden (p = 0.026) in obese mice. In addition to these tumor effects, VEGFR-1 inhibition reduced weight gain, but caused metabolic disorder-hyperinsulinemia-during obesity. Combining metformin with VEGFR-1 inhibition not only prevented this metabolic alteration, but also by recruiting cytotoxic cells further decreased tumor growth in the PAN02 model (p = 0.047). Conclusion: Inactivation of VEGFR-1 signaling prevents weight gain and obesity-induced acceleration of tumor progression in pancreatic and breast cancer models. Targeting VEGFR-1 signaling axis in combination with an anti-diabetic drug such as metformin might be a considerable cancer therapeutic option in the obese setting. Note: This abstract was not presented at the meeting. Citation Format: Joao Incio, Joshua Tam, Nuh Rahbari, Priya Suboj, Daniel McManus, Shan Chin, Trupti Vardan-Kaur, Ana Batista, Suboj Babycutty, Keehoon Jung, Anna Khachatryan, Masabumi Shibuya, Raquel Soares, Dan Duda, Rakesh K. Jain, Dai Fukumura. Role of VEGFR-1 signaling in obesity-induced tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2015-LB-267

Background: Most breast cancer (BC) patients are overweight or obese at the time of diagnosis. Obesity is associated with increased risk, recurrence, and worse prognosis of BC. It has been shown that obesity associates with worse outcome in metastatic kidney or colon cancer treated with bevacizumab. If and how excess body weight contributes to the failure of anti-VEGF therapy in BC is unknown. Results: Here we found that diet-induced obesity promoted resistance to anti-VEGF therapy in two syngeneic mouse breast cancer models. The effects of anti-VEGF therapy on tumor growth and metastasis, VEGF downstream signaling pathways and vessel density were significantly attenuated in obese mice. Under obesity condition, intra-tumor adipocytes increased. These adipocyte-rich regions in breast cancers were hypoxic and overexpress IL-6 or FGF-2 by adipocytes, fibroblasts, and myeloid cells. In IL-6 overexpressing obese breast cancer model (E0771), neutralization of IL-6, either genetically or pharmacologically, abrogated the obesity-induced resistance to anti-VEGF therapy seen in both primary and metastasis sites. This occurred due to a reversion of the obesity-augmented STAT3 signaling and cell proliferation, of hypoxia via vessel normalization, and of immunosuppression. In another breast cancer model (MCaIV), which overexpress FGF-2 under obesity, anti-FGF receptor antibody restored tumor sensitivity to anti-VEGF treatment in obesity. Conclusion: Our findings indicate that obesity promotes resistance to anti-VEGF therapy in breast cancer via the production of pro-inflammatory and angiogenic factors that circumvent the loss of VEGF signaling. Citation Format: Joao Incio, Daniel McManus, Priya Suboj, Nuh Rahbari, Shan M. Chin, Suboj Babycutty, Trupti Vardan-Kaur, Yuhui Huang, Keehoon Jung, Dan Duda, Raquel Soares, Dai Fukumura, Rakesh K. Jain. Obesity promotes resistance to anti-VEGF therapy in breast cancer via pro-inflammatory and angiogenic pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-203. doi:10.1158/1538-7445.AM2015-LB-203