KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Mouse anti-β-actin | Sigma-Aldrich | Cat# A1978, RRID: AB_476692 |
| Rabbit anti-aPKCs | Abcam | Cat# ab59364; RRID: AB_944858 |
| Rabbit anti-CD8 | Abcam | Cat# ab209775; RRID: AB_2860566 |
| Rat anti-CD8 | BD Pharmingen | Cat# 557654, RRID: AB_396769 |
| Rabbit anti-CD19 | Cell Signaling Technology | Cat# 90176S, RRID:AB_2800152 |
| Rabbit anti-MRP8 | Abcam | Cat# ab92331, RRID: AB_2050283 |
| Rabbit anti-CD138 | Thermo Fisher Scientific | Cat# PA5-16918, RRID: AB_10979011 |
| Rabbit anti-FOXP3 | Cell Signaling Technology | Cat#12653, RRID: AB_2797979 |
| Rabbit anti-PDL1 | Cell Signaling Technology | Cat# 64988, RRID: AB_2799672 |
| Rat anti-CD45 | BD Parmingen | Cat# 550539, RRID: AB_2174426 |
| Mouse anti-panCK | Thermo Fisher Scientific | Cat# MA5-13156, RRID: AB_2174426 |
| Rabbit anti-panCK | Abcam | Cat# ab9377, RRID: AB_307222 |
| Rabbit anti-Ki67 | Cell Signaling Technology | Cat#12202, RRID: AB_2620142 |
| Rabbit anti-MUC2 | Abcam | Cat# ab272692, RRID: AB_2888616 |
| Rabbit anti-MUC5AC | Cell Signaling Technology | Cat#61193, RRID: RRID:AB_2799603 |
| Rabbit anti-ANXA10 | Abcam | Cat# ab213656, RRID: RRID:AB_2921231 |
| Rabbit anti-CLU | Abcam | Cat# ab184100, RRID: AB_2892532 |
| Goat anti-CLU | R&D systems | Cat# AF2747, RRID: AB_2083314 |
| Rabbit anti-PDGFRa | Abcam | Cat# ab203491, RRID: AB_2892065 |
| Mouse anti-PDGFRb | Abcam | Cat#ab69506, RRID:AB_1269704 |
| Rabbit anti-CD34 | Abcam | Cat# ab81289, RRID: AB_1640331 |
| Mouse anti-CD31 | Thermo Fisher Scientific | Cat# RB10333P1, RRID: AB_720501 |
| Rat anti-CD31 | Dianova | Cat#DIA-310, RRID:AB_2631039 |
| Rabbit anti-NRG1 | Abcam | Cat# ab191139, RRID: RRID:AB_2921232 |
| Mouse anti-Smooth muscle actin | Dako | Cat# M0851, RRID: AB_2223500 |
| Biotinylated recombinant HA-binding protein (HTI-601) | Halozyme | Provided by Halozyme Therapeutics |
| Goat anti-Mouse IgG1, secondary, HRP | Thermo Fisher Scientific | Cat# PA1-74421, RRID: AB_10988195 |
| Goat anti-Mouse IgG, secondary, HRP | Thermo Fisher Scientific | Cat# 31430, RRID:AB_228307 |
| Goat anti-Rabbit IgG, secondary, HRP | Thermo Fisher Scientific | Cat# 31461, RRID: AB_228347 |
| Goat anti-Rat IgG, secondary, HRP | Thermo Fisher Scientific | Cat# 62-9520, RRID: AB_87993 |
| Donkey anti-Rabbit IgG, secondary, Alexa Fluor 568 | Thermo Fisher Scientific | Cat# A10042, RRID: AB_2534017 |
| Goat anti-Rabbit IgG, secondary, IRDye 800 | LI-COR Biosciences | Cat# 926-32211, RRID: AB_621843 |
| Goat anti-Mouse IgG1, secondary, IRDye 800 | LI-COR Biosciences | Cat# 926-32350, RRID: AB_2782997 |
| Goat anti-Mouse IgG, secondary, IRDye 800 | LI-COR Biosciences | Cat# 926-32210, RRID: AB_621842 |
| InVivoMAb rat IgG2b isotype control | BioXCell | BE0090, RRID:AB_1107780 |
| InVivoMAb anti-mouse PD-L1 (B7-H1) | BioXCell | BE0101, RRID:AB_10949073 |
| InVivoPlus anti-mouse CTLA-4 (CD152) | BioXCell | BP0164, RRID:AB_10949609 |
| Biological samples | ||
| Human Colorectal cancers (CRC) | Osaka City University Hospital, Osaka, JAPAN | N/A |
| Chemicals, peptides, and recombinant proteins | ||
| HBSS (no calcium, no magnesium) | GIBCO | Cat# 14175095 |
| Dimethyl Sulfoxide | Fisher BioReagents | Cat# BP2311 |
| Chloroform | Sigma-Aldrich | Cat# 288306 |
| Methanol | Sigma-Aldrich | Cat# 1424109 |
| Tryple Express | Life Technologies | Cat# 12605-010 |
| HEPES | Gibco | Cat# 15630080 |
| PBS (no calcium, no magnesium) | Gibco | Cat# 10010-023 |
| Quick-RNA Miniprep Kit | Zymo Research | Cat# R1054 |
| RNAlater Stabilization Solution | Invitrogen | Cat# AM7021 |
| TRIzol | Thermo Fisher Scientific | Cat# 15596018 |
| Advanced DMEM/F12 | Thermo Fisher Scientific | Cat# 12634010 |
| RPMI | Corning | Cat#15-040-CV |
| GlutaMAX Supplement | Thermo Fisher Scientific | Cat# 35050061 |
| Penicillin-Streptomycin solution | Corning | Cat# 30-002-CI |
| Y-27632 | Tocris | Cat# 1254 |
| B27 Supplement | Thermo Fisher Scientific | Cat# 17504001 |
| B27 Supplement minus vitamin A | Thermo Fisher Scientific | Cat# 12587001 |
| N2 Supplement | Thermo Fisher Scientific | Cat# 17502048 |
| Murine EGF | Thermo Fisher Scientific | Cat# PMG8045 |
| Human EGF | Peprotech | Cat#AF-100-15 |
| Human FGF-2 | Peprotech | Cat#100-18B |
| Human IGF-1 | Biolegend | Cat#590906 |
| Recombinant human Wnt-3a | R&D systems | Cat#5036-WN/CF |
| A83-01 | Sigma-Aldrich | Cat#SML078 |
| Human [Leu15]-Gastrin I | Sigma-Aldrich | Cat# G9145 |
| Recombinant murine R-Spondin 1 | R&D systems | Cat# 3474-RS-050 |
| Recombinant murine Noggin | Peprotech | Cat# 250-38 |
| Recombinant murine M-CSF | Peprotech | Cat# 315-02 |
| Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | Cat# 356230 |
| Liberase™ TM Research Grade | Sigma-Aldrich | Cat# 5401119001 |
| Collagenase A | Sigma-Aldrich | Cat# 10103586001 |
| Dispase II | Sigma-Aldrich | Cat# #D4693 |
| DNase I | Sigma-Aldrich | Cat# 04716728001 |
| 4-OH-tamoxifen | Millipore-Sigma | Cat# H7904 |
| TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | Cat# A36498 |
| DAPI | Life Technologies | Cat# D1306 |
| Streptavidin, secondary, Alexa Fluor 568 | Thermo Fisher Scientific | Cat# S11226 |
| Streptavidin, secondary, Alexa Fluor 488 | Thermo Fisher Scientific | Cat# S11223 |
| Trypan Blue Solution, 0.4% | Gibco | Cat# 15250061 |
| Critical commercial assays | ||
| Neon™ 10μl Electroporation Kit | Thermo Fisher Scientific | Cat# MPK1096 |
| Chromium Single Cell 3′ Gene Expression Solution v2 | 10X Genomics | Cat#PN-120237 |
| EasySep™ Dead Cell Removal (Annexin V) Kit | STEMCELL Technologies | Cat# 17899 |
| VECTASTAIN® Elite® ABC-HRP Kit | Vector | Cat# PK-6100 |
| Automated Opal 7-Color IHC Kit | AKOYA BIOSCIENCES | Cat# NEL821001KT |
| Manual Opal 4-Color IHC Kit | AKOYA BIOSCIENCES | Cat# NEL810001KT |
| Trichrome Stain (Masson) Kit | Sigma-Aldrich | Cat# HT15 |
| Corning Biocoat Control Inserts | Corning | Cat# 354578 |
| Alexa Fluor™ 488 Tyramide Superboost™ Kit | Thermo Fisher Scientific | Cat# B40932 |
| Deposited data | ||
| sc-RNA-seq | This study | GEO: GSE207780 |
| 3’ RNA-seq (Prkcifl/fl Prkczfl/fl Villin-Cre-Veh or -PEGPH20 treated) | This study | GEO: GSE207778 |
| 3’ RNA-seq (sgPrkci/Prkcz and sgC MTOs) | This study | GEO: GSE207776 |
| 3’ RNA-seq (sgPrkci/Prkcz MTOs-Veh or -PEGPH20 treated) | This study | GEO: GSE207779 |
| Raw Data | This study; Mendeley Data | https://doi.org/10.17632/vbzbzhrw98.1 |
| Single cell 3′ RNA sequencing from patients with CRC | Lee et al.26 | GEO: GSE132465 |
| Single cell 3′ RNA sequencing from patients with CRC | Uhlitz et al.40 | GEO: GSE166555 |
| Single cell 3′ RNA sequencing from patients with CRC | Pelka et al.41 | GEO: GSE178341 |
| Microarray from primary CRC tumors | Jorissen et al.73 | GEO: GSE14333 |
| Microarray from primary CRC tumors | Marisa et al.74 | GEO: GSE39582 |
| TCGA-COREAD | cBioportal | http://www.cbioportal.org/index.do |
| Experimental models: Cell lines | ||
| Human CRC (sgPRKCI/PRKCZ) | This study | N/A |
| Mouse tumor organoid (MTO) | Tauriello et al.17 | N/A |
| Experimental models: Organisms/strains | ||
| Mouse: Prkcifl/fl Prkczfl/fl | Nakanishi et al.13 | N/A |
| C57BL/6J | The Jackson Laboratory | Stock No: 000664 |
| Oligonucleotides | ||
| Real-time PCR primers | This manuscript | Table S1 |
| CRISPR guides | This manuscript | Table S2 |
| Software and algorithms | ||
| Graphpad Prism 8 | Graphpad | https://www.graphpad.com/scientificsoftware/ |
| QuPath v.0.1.3 | Queen’s University, Belfast, Northern Ireland | https://qupath.github.io |
| ImageJ | NIH | https://imagej.nih.gov/ij/ |
| Zen blue | Zeiss | https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html |
| RStudio (1.1.456) | R Core Team | https://www.r-project.org/ |
| R | R Core Team | https://www.r-project.org/ |
| GSVA; version 1.26.0 | Bioconductor | https://github.com/rcastelo/GSVA |
| GSEA (v4.1.0) | Broad Institute | http://www.broadinstitute.org/gsea/index.jsp |
| BaseSpace | Illumina | https://basespace.illumina.com/ |
| Morpheus | Broad Institute | https://software.broadinstitute.org/morpheus/ |
| CMScaller v0.99.1 | Eide et al.72 | https://github.com/peterawe/CMScaller |
| CellPhoneDB v.2.0.0 | Efremova et al.43 | https://www.cellphonedb.org |
| GenePattern | Broad Institute | https://cloud.genepattern.org/gp/pages/login.jsf |
| Lexogen QuantSeq DE 1.3.0 | BlueBee Cloud | https://www.bluebee.com |
| Cell Ranger (v3.0) | Langmead et al.75 10X genomics | http://software.10xgenomics.com/single-cell/overview/welcome |
| Seurat 3.0 | Stuart et al.76 | https://github.com/Satijalab/seurat |
| kb_python | Bray et al.69;Melsted et al.70 | https://github.com/pachterlab/kb_python |
| scVelo package | Bergen et al.71 | https://scvelo.readthedocs.io/VelocityBasics/ |
| Harmony | Korsunsky et al.67 | https://portals.broadinstitute.org/harmony/articles/quickstart.html |
| PhenoChart Whole Slide Viewer | AKOYA BIOSCIENCES | https://www.akoyabio.com/support/software/phenochart-whole-slide-viewer/ |
| CaseViewer | 3DHistech, Budapest, Hungary | https://www.3dhistech.com/solutions/caseviewer/ |
| Other | ||
| EVOS FL Auto Imaging System | Thermo Fisher Scientific | N/A |
| EVOS M5000 Imaging System | Thermo Fisher Scientific | N/A |
| TissueLyser II | QIAGEN | Cat# 85300 |
| NanoDrop 1000 spectrophotometer | Thermo Fisher Scientific | N/A |
| Zeiss LSM 710 NLO Confocal Microscope | Zeiss | N/A |
| Neon™ Transfection System | Thermo Fisher Scientific | Cat# MPK5000 |
| Pannoramic Scanner | 3DHistech, Budapest, Hungary | N/A |
| Vectra Polaris Automated Quantitative Pathology Multispectral Imaging System | AKOYA BIOSCIENCES | N/A |