Antiangiogenic immunotherapy suppresses desmoplastic and chemoresistant intestinal tumors in mice
Simone Ragusa, … , Michele De Palma, Tatiana V. Petrova
Simone Ragusa, … , Michele De Palma, Tatiana V. Petrova
Published February 4, 2020
Citation Information: J Clin Invest. 2020;130(3):1199-1216. https://doi.org/10.1172/JCI129558.
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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 3

Prox1 loss generates desmoplastic tumors in the presence of activated Kras.

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Prox1 loss generates desmoplastic tumors in the presence of activated K...
(A) Reduced clonogenic capacity of Prox1–/– intestinal stem cells. Apcfl/fl KrasLSL-G12D Tp53fl/fl villin-CreERT2 (AKP) and Apcfl/fl KrasLSL-G12D Tp53fl/fl Prox1fl/fl villin-CreERT2 (AKPP) organoids (n = 12 per genotype). (B) AKP and AKPP organoids. Upper row shows Matrigel disks with organoids. Scale bars: 1.2 mm. Lower row shows H&E-stained organoid sections. Scale bars: 20 μm. Arrow indicates epithelial polarization loss. (C) WNT signaling and differentiation in AKP and AKPP organoids. Graph shows qRT-PCR for the indicated genes, and data are presented in a heatmap (n = 5). (D) PROX1 loss delayed tumor development. Mice were injected s.c. with 50 AKP or AKPP organoids. AKP, n = 5; AKPP, n = 6. (E) Masson’s trichrome staining of advanced tumors. Scale bars: 50 μm. (F) Increased stromal reaction and invasion in AKPP tumor margins. Images in top row show staining for PROX1 (green), E-cadherin (red), Ki67 (white), and DNA (blue). Images in bottom row show staining for POSTN (green), CD44V6 (red), α-SMA (white), and DNA (blue). Scale bars: 50 μm. (G) AKPP organoids produced profibrotic factors. Heatmap of the indicated genes (n = 5). (H) AKPP organoids promoted fibroblast proliferation. Ki67 and Ccnb1 expression by qRT-PCR. Data were normalized to the control mean (n = 6). (I) Tumor growth curves and weights. Mice were implanted with 50 AKP or 300 AKPP organoids ± 5000 tdTomato+ intestinal fibroblasts (Fibro.) (n = 7). (J) AKPP cells sustained fibroblasts. Images show control and tdTomato+ (red) fibroblast–coinjected tumors. Scale bars: 2 mm. (K) Fibroblast coimplantation effects on tumor desmoplasia, PROX1 expression, and cell proliferation. Images show staining for PROX1 (green), α-SMA (red), Ki67 (white), and DNA (blue). Scale bars: 50 μm. (L) Quantification of the data shown in K. α-SMA+ and Ki67+ areas were normalized to the total tumor area and the AKP mean. (M) Fibroblast and tissue stiffness effect on organoids. Organoids with or without tdTomato+ fibroblasts in hydrogels with normal mucosa (1.3 kPa) or tumor (2.7 kPa) stiffness. Scale bars: 1.2 mm. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by Student’s t test (A and C) or 1-way (H and L) or 2-way (I) ANOVA with Tukey’s multiple comparisons test, scatterplot, or mean ± SD.