Drug profiling in an immune cell-tumor spheroid co-culture model

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1 Oncology Drug Discovery Molecular Pharmacology Drug profiling in an immune cell-tumor spheroid co-culture model Silvia Goldoni, Investigator Novartis Institutes for Biomedical Research, Cambridge MA, USA 3D Tissue Models 2017

2 Outline of presentation Short introduction on 3D cultures for immuno-oncology studies Overview of our co-culture platform and characterization Immune profiling of a Novartis targeted therapy Effort to generate complex in vitro models Planned drug discovery efforts 2

3 Why 3D for immune profiling? Multicellular spheroids have enhanced immune suppressive phenotype compared to 2D tumor cell cultures : Lactate production Immune suppressive protein expression (IL8, VEGF, FasL) Limited antigen presentation Less sensitivity to IFNγ More physiological immune synapse Opportunity to study immune cell infiltration Cancer cells PBMCs 3

4 Co-culture overview Day -1: Tumor cell line seeding Day 0: add fresh PBMCs (CFSE-labeled) + treatment +/- stimulation 200um 100um Cells spontaneously gather in each micro-space poly-hema coating (low adherence surface) e.g. OvCar-8 OvCar8-mcherry PBMC Immune cell infiltration Cytokine release Tumor cell killing PBMC proliferation Day 5: data collection 4

5 Co-culture characterization T-cell activation: proliferation Immune cells- no tumor spheroid Immune cells- tumor associated Tumor spheroid size Proliferation Immune cell proliferation is not affected by soluble factors in the tumor line conditioned medium 5

6 T N F a [p g /m l] R A N T E S [p g /m l] IF N g [p g /m l] G M C S F [p g /m l] Co-culture characterization T-cell activation: cytokine release E lp la s ia P la te A lo n e T C e lls + S m a ll S p h e r e s T C e lls + M e d iu m S p h e r e s T C e lls + L a r g e S p h e r e s 0 E lp la s ia P la te A lo n e T C e lls + S m a ll S p h e r e s T C e lls + M e d iu m S p h e r e s T C e lls + L a r g e S p h e r e s E lp la s ia P la te A lo n e T C e lls + S m a ll S p h e r e s T C e lls + M e d iu m S p h e r e s T C e lls + L a r g e S p h e r e s 0 E lp la s ia P la te A lo n e T C e lls + S m a ll S p h e r e s T C e lls + M e d iu m S p h e r e s T C e lls + L a r g e S p h e r e s 6

7 Co-culture characterization Immune suppressive features Nat. Immunol IDO suppresses T-cell function promoting tumor immune tolerance 7

8 OvCar8 + PBMC OvCar8 d4 Co-culture characterization Immune suppressive features IDO1* ISH TGF-β ISH *Confirmed by qpcr 8

9 Immune profiling of a Novartis targeted therapy Testing the co-culture platform

10 Therapy enhances immune cell proliferation DMSO 0.5 µm 1 µm 10 µm Unstimulated Anti-CD3 stimulated* *Similar results observed in 4 of 5 donors tested CFSE= Carboxyfluorescein succinimidyl ester 10

11 Therapy enhances immune cell proliferation in co-culture DMSO Inactive scaffold (10 µm) Treatment* (10 µm) % proliferating cells *larger spheroids similar result with 1 µm 11

12 OvCar8 + PBMC OvCar8 d4 IDO1 expression is inhibited by treatment IDO1* ISH Treatment Co-culture 12

13 % Control Does treatment enhance immune cell-mediated tumor cell killing? Inactive scaffold Tool compound Treatment Concentration Concentration Concentration *Viability assay using CTG 3D at day 6 13

14 Absolute tumor cell counts Absolute tumor cell counts Treatment enhances immune cellmediated tumor cell killing k tumor cell counts 10k tumor cell counts unstimulated sol anti-cd3 unstimulated sol anti-cd DMSO 1uM MHP982 20uM LMD099 1uM TNO155 DMSO 1uM MHP982 20uM LMD099 1uM TNO155 DMSO 1uM MHP982 20uM LMD099 1uM TNO155 E Tumor only DMSO 1uM MHP982 20uM LMD099 1uM TNO155 DMSO 1uM MHP982 20uM LMD099 1uM TNO155 DMSO 1uM MHP982 20uM LMD099 1uM TNO155 Tumor-PBMC coculture E Tumor only E Co-culture Tumor-PBMC coculture Tumor only DMSO 1uM MHP982 20uM LMD099 1uM TNO155 DMSO 1uM MHP982 20uM LMD099 1uM TNO155

15 Chemoattractant cytokine IP10 is upregulated by treatment Co-culture PBMC + anti-cd3 Tumor spheroids Produced by monocytes, endothelial cells, fibroblasts in response to IFNγ and other cytokines Chemoattractant for anti-tumoral leukocytes (T, monocytes, NK cells) via interaction with CXCR3 15

16 CD45- CD45+ What cell types express IP10? PBMC Co-culture+DMSO Co-culture+DMSO +anti-cd3 Co-culture +anti-cd3 +Inactive 1 µm Co-culture +anti-cd3 +Treatment 1 µm Tumor cells Tumor cells + Treatment 1 µm Co-culture +DMSO Co-culture+DMSO +anti-cd3 Co-culture +anti-cd3 +Inactive 1 µm Co-culture +anti-cd3 +Treatment 1 µm 16

17 Treatment modulates secretion of IL8 and GM-CSF 17 Co-culture IL8: Supports proliferation, survival and migration of cancer cells Associated with tumor-induced immune suppression Co-culture GM-CSF: Adjuvant in cancer vaccines: boosts antigen presentation by dendritic cells Generally considered immune stimulatory Context specific immune suppressive role in the tumor microenvironment

18 Do immune cells infiltrate? Tumor spheroids: Histology Light-sheet microscopy High content imaging ECM-embedded tumor cultures 3D bioprinting PE Opera Phenix 18

19 Treatment enhances recruitment of CD3+ cells to tumor spheroids OvCAR8/PBMC H&E 20x OvCAR8/PBMC CD3 20x OvCAR8/PBMC/Treatment H&E 20x OvCAR8/PBMC/Treatment CD3 20x 19

20 Treatment stimulates PBMC migration through tumors No treatment Treatment 10 µm 20

21 Pathology relevance of modeling tumors in vitro with ECM and stroma 21

22 Our approach along side traditional methods: light-based bio-printing 1. Mix cells + lightresponsive biomatrix 2. Apply light-mask to polymerize biomatrix at micrometer scale 3. Wash out free cells; a precise cellular construct is obtained 22

23 PBMC migrate through a novel pathology relevant bioink Day1 Day2 Day3 OvCar8-mCherry PBMC 20x magnification Maximal projection 23

24 On going drug discovery efforts Expanding co-culture from a semi-automated to a fully automated assay Profiling pre-clinical candidates and clinical therapies in co-culture using different dosing strategies Evaluating phenotype of anti-pd1 therapy and combinations Developing an imaging-based immune cell infiltration assay 24

25 Acknowledgements Oncology Steve Kovats Ye Wang Bhavesh Pant Tamara Gilbert LeighAnn Alexander Lesley Griner Serena Silver Shumei Liu Kathy Hsiao Ying-Nan Chen Immuno-oncology Wei Guan Beatriz Perez Bill Hastings Glenn Dranoff Analytical Sciences &Imaging Christina Dornelas Farid SariSarraf Ned Kirkpatrick Ophthalmology Xin Qu Arnaud Lacoste Preclinical Safety Joyce Judge Keith Mansfield 25

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