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 7

A2V+aCD40 antitumor activity is T cell dependent.

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A2V+aCD40 antitumor activity is T cell dependent. (A) Tumors were treate...
(A) Tumors were treated with control mAbs, A2V+aCD40, or A2V+aCD40+α-CD8+α-CD4. Quantification of intratumoral T cells was determined by the fold change versus the AP IgGs mean. AP IgGs, n = 8; AP A2V+aCD40 AP A2V+aCD40+αCD8+αCD4 or APP A2V+aCD40, n = 9; APP IgGs, n = 10; APP A2V+aCD40+αCD8+αCD4, n = 7. (B) FACS analysis of lymph node CD11b+CD11c+CD86+ DCs and CD11b–B220–MHCII–CD8+ T cells and splenic CD11b–B220–MHCII–CD8a+CD44+CD62l+ Tm cells. (C) Quantification of intratumoral cytotoxicity. Quantification of CD11b–CD3+CD8a+IFN-γ+ and GZMB+ T cells, percentage of CD45+ cells, and qRT-PCR analysis of Prf1 and Gzma expression, normalized to the AP of IgGs mean. (D) T cells mediated cancer cell death in A2V+aCD40-treated tumors. Cell death was determined by the percentage of necrotic glands. PH3+E-cadherin+ cells were normalized to the AP IgGs mean. (E) Effect of T cell depletion on tumor cell death and stroma. Images show staining for PH3 (green), α-SMA (red), E-cadherin (white), and DNA (blue). Scale bars: 50 μm. (F) Quantification of vessel density and mural coverage. Data were normalized to the AP IgGs mean. (G) A2V+aCD40 reduced stromal content after T cell depletion. Quantification of stromal content and proliferation is shown, and data were normalized to the AP IgGs mean. (H) Comparison of A2V+aCD40 versus A2V+B20 combination treatment. Images show staining for CD8a (green), VE-cadherin (red), E-cadherin (white), and DNA (blue). AP IgGs, n = 10; AP B20+aCD40 or AP A2V+aCD40, n = 11; APP IgGs or APP B20+aCD40, n = 8; APP A2V+aCD40, n = 9. Scale bars: 50 μm. (I) Quantification of CD8+ T cell infiltration and cell death. Cell death was determined by the percentage of necrotic tumor glands. CD8a+ T cell infiltration was determined by the fold change versus the AP IgGs mean. (J) PROX1hi tumors display high WNT signaling, a small amount of stroma, and are chemosensitive. Low PROX1 levels blunt WNT activation and promote tumor desmoplasia, angiogenesis, and chemoresistance. A2V+aCD40 normalizes the tumor vasculature, reduces Treg numbers, expands TLSs, and induces antitumor responses. 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. Comb., combination; Tdepl., T cell depletion.