Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG
- Physical Sciences Oncology Center at Penn, University of Pennsylvania, United States
- Molecular and Cell Biophysics Lab, University of Pennsylvania, United States
- Bioengineering Graduate Group, University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
MPS1 kinase inhibition (MPS1i)-induced chromosomal instability generates microenvironment conditions that skew toward anti-cancer M1-like macrophages relative to a pro-cancer M2-like phenotype.
(A) Schematic for treatment of B16F10 mouse melanoma cells with MPS1 inhibitors (MPS1i), such as reversine, or the equivalent volume of DMSO vehicle control. After 24 hr, cells were washed twice …
Figure 1—figure supplement 1
Characterization of MPS1 kinase inhibition (MPS1i)-induced genome and chromosomal instability in B16F10 mouse melanoma.
(A) Quantification of micronuclei induced by different MPS1 inhibitors (AZ3146, BAY 1217389, and reversine) at different concentrations. Statistical significance was calculated by ordinary one-way …
Figure 2 with 4 supplements
Chromosomally unstable tumoroids and tumors in early stages show increased anti-cancer macrophage polarization and activity.
(A) Schematic for ‘immuno-tumoroids’. Tumoroids are formed with B16F10 cells on non-adhesive, U-bottom-shaped wells and after pre-treating with either MPS1 kinase inhibition (MPS1i) (reversine) or …
Figure 2—figure supplement 1
Characterization of MPS1 kinase inhibition (MPS1i)-induced genome and chromosomal instability in B16F10 mouse melanoma.
(A) Flow cytometry analysis of CD47 and Tyrp1 expression on B16F10 mouse melanoma cells. Representative histograms for anti-CD47 (left) and anti-Tyrp1 (right) binding to B16F10 CD47 knockout (KO) …
Figure 2—figure supplement 2
Macrophages readily clear chromosomal instability (CIN)-afflicted tumoroids but only if CIN is accompanied by proliferation deficits.
(A) Timeline and schematic for generating engineered ‘immuno-tumoroids’ for time-lapsed studies of macrophage-mediated phagocytosis of cancer cells. Tumoroids are formed by plating and culturing …
Figure 2—figure supplement 3
Flow cytometry gating strategy for identification and quantification of macrophage infiltrate and characterization in chromosomal instability (CIN)-afflicted and chromosomally stable B16F10 tumors.
Representative flow cytometry gating strategy for in vivo B16F10 CD47 knockout (KO) tumor immune infiltrate five days after initial challenge. Tumors were comprised of cells treated with either (A) …
Figure 2—figure supplement 4
MPS1 kinase inhibition (MPS1i)-induced chromosomal instability upregulates MHC-1 class molecules on B16F10, suggesting increased antigen presentation.
Flow cytometry analysis and quantification of H-2Kb expression on B16F10 mouse melanoma cells. B16F10 cells were treated with MPS1i or the equivalent volume of DMSO vehicle control. Concentrations …
Figure 3 with 1 supplement
MPS1 kinase inhibition (MPS1i)-induced chromosomally unstable cancer cells are maximally cleared when IgG-opsonized and depleted of the CD47 macrophage checkpoint.
(A) Timeline for in vitro treatment of B16F10 cells prior to injection in mice and then subsequent therapeutic treatment for tumor-challenged mice. Prior to tumor inoculation, B16F10 CD47 knockout …
Figure 3—figure supplement 1
MPS1 kinase inhibition (MPS1i)-induced chromosomal instability (CIN) favors clearance when paired with CD47 knockout (KO) and IgG opsonization, regardless of the degree of CIN.
Tumor growth curve of projected tumor area versus days after tumor challenge, with B16F10 CD47 KO cells. Each line represents a separate tumor and is fit with an exponential growth equation: A = A0ek…
Figure 4 with 3 supplements
MPS1 kinase inhibition (MPS1i)-induced chromosomal instability favors induction of pro-phagocytic de novo IgG and can lead to durable acquired immunity.
(A) Schematic for sera collection from surviving mice from Figure 3C and D and follow-up experiments to characterize any de novo anti-cancer IgG antibodies and their functionality in vitro and in …
Figure 4—figure supplement 1
Survivors challenged with chromosomal instability (CIN)-afflicted tumors generate anti-cancer IgG, regardless of the degree of CIN.
(A) Median fluorescence intensity quantification of IgG2a/c and IgG2b binding from sera from surviving mice with different degrees of CIN by titrating reversine concentration used to treat B16F10 …
Figure 4—figure supplement 2
Flow cytometry gating strategy for identification and quantification of immune infiltrate and characterization in re-challenge experiments.
Representative flow cytometry gating strategy for in vivo B16F10 CD47 knockout (KO) tumor immune infiltrate from second challenge non-survivors and age-matched naïve controls. Singlets were …
Figure 4—figure supplement 3
Growth of chromosomal instability (CIN)-afflicted wild-type (WT) tumors in T- and B-cell deficient mice and T- and B-cell replete mice.
Similar growth delays are found for MPS1 kinase inhibition (MPS1i)-pre-treated B16F10 cells in T- and B-cell deficient NSG mice and immunocompetent C57BL/6 mice. Both types of mice have functional …
Author response image 1
Author response image 2
Author response image 3
Author response image 4
Growth of CIN-afflicted wild-type (WT) tumors in T- and B-cell deficient mice and T- and B-cell replete mice.
Similar growth delays for MPS1i-pretreated B16F10 cells in T- and B-cell deficient NSG mice and immunocompetent C57BL/6 mice. Both types of mice have functional macrophages. Parallel studies in vivo …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background Mus musculus | C57BL/6J | Jackson Laboratory | Stock# 000664; RRID:IMSR_JAX:000664 | Sex: Male (because B16 cell line is male derived) |
| Strain, strain background Mus musculus (male) | NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ | Perelman School of Medicine Stem Cell Xenograft Core | RRID:SCR_010035 | Sex: Male |
| Cell line (Mus musculus) | B16F10 | ATCC | Cat# CRL-6475; RRID:CVCL_0159 | Mouse melanoma |
| Cell line (Mus musculus) | B16F10 CD47 KO | PMID: 31964705 | CD47 KO | Hayes et al., 2020 |
| Cell line (Mus musculus) | B16F10 sgCtrl | PMID: 31964705 | sgCtrl | Hayes et al., 2020 |
| Biological sample (Mus musculus) | C57BL/6J bone marrow cells | This paper | Flushed from femurs, tibias; differentiation into bone marrow-derived macrophages (BMDMs) or adoptive transfer | |
| Biological sample (Mus musculus) | C57BL/6J serum | This paper | Retro-orbital blood collected from convalescent or naïve mice, clotted for 1 hr, separated by centrifugation | |
| Antibody | InVivoMab mouse anti-human/mouse Tryp1 (clone TA99) | BioXCell | Cat# BE0151; RRID:AB_10949462 | (1:1000) Opsonization (10–20 μg/mL) In vivo (250 μg/100 μL per dose) |
| Antibody | InVivoMab mouse IgG2a isotype control (clone C1.18.4) | BioXCell | Cat# BE0085; RRID:AB_1107771 | (1:1000) (10–20 μg/mL) In vivo (250 μg/100 μL per dose) |
| Antibody | Ultra-LEAF rat anti-mouse SIRPα (clone P84) | BioLegend | Cat# 144036; RRID:AB_2832517 | Blockade (18 μg/mL=1:50) |
| Antibody | InVivoMab rat anti-mouse CD47 (clone MIAP301) | BioXCell | Cat# BE0270; RRID:AB_2687793 | (1:500) Flow cytom. (20 μg/mL) |
| Antibody | Alexa Fluor 647 anti-mouse IgG [H+L] (donkey polyclonal) | Thermo Fisher Invitrogen | Cat# A-31571; RRID:AB_162542 | Flow cytom. (10 μg/mL=1:200) |
| Antibody | Alexa Fluor 647 anti-rat IgG [H+L] (goat polyclonal) | Thermo Fisher Invitrogen | Cat# A-21247; RRID:AB_141778 | Flow cytom. (10 μg/mL=1:200) |
| Antibody | Horse radish peroxidase sheep anti-mouse [H+L], polyclonal | GE Life Sciences | Cat# NA931V | WB (1:500) |
| Antibody | Mouse anti-β-actin (clone C4) | Santa Cruz | Cat# sc47778; RRID:AB_626632 | WB (1:1000) |
| Antibody | Brilliant Violet 650 rat anti-mouse CD45 (clone 30-F11) | BioLegend | Cat# 103151; RRID:AB_2565884 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | Brilliant Violet 785 rat anti-mouse CD45 (clone 30-F11) | BioLegend | Cat# 103149; RRID:AB_2564590 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | APC/Cyanine7 rat anti-mouse CD45 (clone 30-F11) | BioLegend | Cat# 103115; RRID:AB_312980 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | APC rat anti-mouse/human CD11b (clone M1/70) | BioLegend | Cat# 101212; RRID:AB_312795 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | PE/Cyanine7 rat anti-mouse/human CD11b (clone M1/70) | BioLegend | Cat# 101216; RRID:AB_312799 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | PE/Dazzle 594 rat anti-mouse Ly6G (clone 1A8) | BioLegend | Cat# 127647; RRID:AB_2566318 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | PerCP rat anti-mouse Ly6G (clone 1A8) | BioLegend | Cat# 127653; RRID:AB_2616998 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | PE rat anti-mouse F4/80 (clone BM8) | BioLegend | Cat# 123110; RRID:AB_893486 | Flow cytom. (10 µg/mL=1:20) |
| Antibody | Brilliant Violet 605 rat anti-mouse Ly-6C (clone HK1.4) | BioLegend | Cat# 128035 RRID:AB_2562352 | Flow cytom (5 µL/100 µL=1:20) |
| Antibody | APC rat anti-mouse I-A/I-E (clone M5/114.15.2) | BioLegend | Cat# 107614; RRID:AB_313329 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | Pacific Blue rat anti-mouse CD86 (clone GL-1) | BioLegend | Cat# 105022; RRID:AB_493466 | Flow cytom. (10 µg/mL=1:50) |
| Antibody | APC/Cyanine7 rat anti-mouse CD86 (clone GL-1) | BioLegend | Cat# 105029; RRID:AB_2074993 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | APC rat anti-mouse CD206 (clone C068C2) | BioLegend | Cat# 141707; RRID:AB_10896057 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | Brilliant Violet 421 rat anti-mouse CD206 (clone C068C2) | BioLegend | Cat# 141717; RRID:AB_2562232 | Flow cytom. (5 µL/100 µL=1:20) |
| Antibody | PE/Cyanine7 rat anti-mouse CD163 (clone S15049F) | BioLegend | Cat# 156707; RRID:AB_2910324 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | APC/Cyanine7 Armenian hamster anti-mouse CD3e (clone 145-2C11) | BioLegend | Cat# 100329; RRID:AB_1877171 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | Alexa Fluor 647 rat anti-mouse CD8a (clone 53-6.7) | BioLegend | Cat# 100727; RRID:AB_493424 | Flow cytom. (2.5 µg/mL=1:200) |
| Antibody | APC mouse anti-mouse H-2kb/H-2Db (clone 28-8-6) | BioLegend | Cat# 114613; RRID:AB_2750193 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | PE rat anti-mouse IgG2a (clone RMG2a-62) | BioLegend | Cat# 407108; RRID:AB_10549974 | Flow cytom. (5 µg/mL=1:40) |
| Antibody | APC rat anti-mouse IgG2b (clone RMG2b-1) | BioLegend | Cat# 406711; RRID:AB_2750277 | Flow cytom. (2.5 µg/mL=1:80) |
| Antibody | TruStain FcX PLUS rat anti-mouse CD16/32 (clone S17011E) | BioLegend | Cat# 156603; RRID:AB_2783137 | Flow cytom. Fc block (2.5 µg/mL=1:200) |
| Peptide, recombinant protein | Macrophage colony-stimulating factor (M-CSF) | BioLegend | Cat# 576406 | BMDM differentiation (20 ng/mL) |
| Commercial assay or kit | Vybrant CFDA-SE Cell Trace | Thermo Fisher Invitrogen | Cat# V12883 | |
| Commercial assay or kit | CellTracker DeepRed | Thermo Fisher Invitrogen | Cat# C34565 | |
| Commercial assay or kit | NEBNext Ultra II RNA Library Prep Kit | NEB | Cat# E7770S | |
| Sequence-based reagent | NEBNext Multiplex Oligos for Illumina | NEB | Cat# E7335 | |
| Commercial assay or kit | Chromium Single Cell Gene Expression kit | 10x Genomics | Cat# PN-1000128; Cat# PN-1000127; Cat# PN-1000213 | |
| Chemical compound, drug | Reversine | Cayman Chemical | Cat# 10004412 | MPS1 inhibitor |
| Chemical compound, drug | AZ3146 | Cayman Chemical | Cat# 19991 | MPS1 inhibitor |
| Chemical compound, drug | BAY 12-17389 | Selleck Chemicals | Cat# S8215 | MPS1 inhibitor |
| Software, algorithm | Prism v9.4 | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | FCS Express 7 | De Novo Software | ||
| Other | Anti-adherence rinsing solution | StemCell Technologies | Cat# 07010 | Surface treatment for tumoroid studies |
| Other | ACK lysing buffer | Thermo Fisher Gibco | Cat# A1049201 | Bone marrow and tumor red blood cell lysis |
| Other | Dispase | Corning | Cat# 354235 | Tumor disaggregation |
| Other | DNAse I | Millipore Sigma | Cat# 101041159001 | Tumor disaggregation |
| Other | Collagenase type IV | Thermo Fisher Gibco | Cat# 17104-019 | Tumor disaggregation (4 mg/mL) |
