Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice
Simone Ragusa, Borja Prat-Luri, Alejandra González-Loyola, Sina Nassiri, Mario Leonardo Squadrito, Alan Guichard, Sabrina Cavin, Nikolce Gjorevski, David Barras, Giancarlo Marra, Matthias P. Lutolf, Jean Perentes, Emily Corse, Roberta Bianchi, Laureline Wetterwald, Jaeryung Kim, Guillermo Oliver, Mauro Delorenzi, Michele De Palma, Tatiana V. Petrova
Simone Ragusa, Borja Prat-Luri, Alejandra González-Loyola, Sina Nassiri, Mario Leonardo Squadrito, Alan Guichard, Sabrina Cavin, Nikolce Gjorevski, David Barras, Giancarlo Marra, Matthias P. Lutolf, Jean Perentes, Emily Corse, Roberta Bianchi, Laureline Wetterwald, Jaeryung Kim, Guillermo Oliver, Mauro Delorenzi, Michele De Palma, Tatiana V. Petrova
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Research Article Angiogenesis Oncology

Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice

Abstract

Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell–mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.

Authors

Simone Ragusa, Borja Prat-Luri, Alejandra González-Loyola, Sina Nassiri, Mario Leonardo Squadrito, Alan Guichard, Sabrina Cavin, Nikolce Gjorevski, David Barras, Giancarlo Marra, Matthias P. Lutolf, Jean Perentes, Emily Corse, Roberta Bianchi, Laureline Wetterwald, Jaeryung Kim, Guillermo Oliver, Mauro Delorenzi, Michele De Palma, Tatiana V. Petrova

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Figure 6

A2V+aCD40 combination reduces AP and APP tumor growth.

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A2V+aCD40 combination reduces AP and APP tumor growth. (A) Treatment sch...
(A) Treatment scheme and weights of tumors in the indicated conditions. AP IgGs, AP A2V, or AP A2V+aCD40, n = 10; AP aCD40, n = 8; APP IgGs, APP A2V or APP A2V+aCD40, n = 7; APP aCD40, n = 6. (B) A2V+aCD40 induced tumor necrosis and stromal destruction. Images show staining for E-cadherin (white), PH3 (green), and α-SMA (red). Scale bars: 50 μm. (C) Quantification of tumor necrosis and proliferation. Necrosis is indicated by the percentage of necrotic glands. Proliferation was quantified by PH3+E-cadherin+ tumor cells normalized to total E-cadherin+ and the AP IgGs mean. (D) A2V and A2V+aCD40 promoted vascular maturation. Images show staining for CD31 (green) and α-SMA (red). Scale bars: 50 μm. (E) Quantification of vessel density, mural coverage, and vessel caliber. Data were normalized to the AP IgGs mean. (F) A2V and A2V+aCD40 reduced desmoplasia. The α-SMA+ stromal area was normalized to the corresponding tumor area and the AP IgGs mean. (G) A2V and A2V+aCD40 effects on stromal proliferation and Mmp14 mRNA. Quantification of PH3+α-SMA+ fibroblasts per tumor area and qRT-PCR for Mmp14 in tumor lysates. Data were normalized to the AP IgGs mean. (H) A2V+aCD40 promoted CD8+ T cells intraepithelial infiltration. Images show staining for CD8a (green), VE-cadherin (red), and E-cadherin (white). Scale bars: 50 μm. (I) CD8+/CD4+ TIL ratio and intraepithelial CD8+ T cell quantification were determined as the fold change versus the AP IgGs mean. (J) A2V+aCD40 increased GZMB+ cytotoxic CD8+ T cells. Intratumoral CD8+GRZB+ T cells were normalized to CD8+ T cells and the AP IgGs mean. (K) A2V and A2V+aCD40 reduced Tregs. Images show staining for Foxp3 (green), CD4 (red), and E-cadherin (white). Scale bars: 50 μm. (L) Quantification of Tregs and CD8+/Treg ratio. CD4+Foxp3+ expression was normalized to CD4+ T cells and the AP IgGs mean. (M) Pathways deregulated by A2V+aCD40, AP A2V+aCD40, or APP A2V+aCD40, n = 4; AP or APP controls, n = 6. FDR <0.05. NES, normalized enrichment scores. Data represent the mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA with Tukey’s multiple comparisons test.