Oregon Health and Science University – 2

Large-scale Characterization of Drug Responses for Clinically Relevant Proteins in Cancer Cell Lines

The study profiles the protein-level responses of a large collection of cancer cell lines to drug perturbations using reverse-phase protein arrays and builds a systematic protein-drug connectivity map.

The results show that integrating perturbed protein responses provides better prediction of drug sensitivity and insights into drug resistance mechanisms and combination therapies.

In Vivo Functional Characterization of EGFR Variants Identifies Novel Drivers of Glioblastoma

In an effort to validate novel functional EGFR variants and pathways, we screen a library of missense mutations of EGFR and found that different variants differentially drive gliomagenesis. Among the differentially activated pathways was lipid metabolism, and perturbation of this pathway delayed glioma-associated death in mouse models.

Experimental Approaches

Tumor bearing mice were generated through in utero electroporation. A construct expressing CRISPR guide sequences that targeted mouse Tp53 and Pten, along with the Cas9 ORF were injected and electroporated into the lateral ventricles of E14.5 mouse brains. Gliomas driven by different EGFR variants were generated by electroporating and piggyBac transposases construct along with a transposable cassette expressing EGFR variants under a constitutively active promoter. When mice demonstrated behaviors characteristic of tumor burden, bulk tumor tissue were dissected. Form these tissue samples, RNA was extracted using Qiagen’s RNeasy Plus Mini Kit. 700ng of RNA was used as template for library preparation using the TruSeq Stranded mRNA LT kit. Using at least 3 biological replicates, pooled libraries were sequenced with approximately 20 million pair-end (2x75) reads per sample using Illumina’s Mid Output v2 kit on a NextSeq500. FASTQ files were quality controlled using fastQC (v0.10.1) and MultiQZ (v0.9), and aligned to the mm10 reference genome.

Tumor-intrinsic SIRPA Promotes Sensitivity to Checkpoint Inhibition Immunotherapy in Melanoma

The study reveals that tumor-intrinsic SIRPA can enhance the sensitivity to anti- PD-1 treatment in melanoma patients, whereas macrophage SIRPA has a well-established role as a major inhibitory modulator in antitumor immunity. The results highlight that the same target in different cell types can have antagonistic effects on immunotherapy.

C5aR1 Inhibition Reprograms Tumor Associated Macrophages and Reverses PARP Inhibitor Resistance in Breast Cancer

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases, including BRCA1/2 mutant breast cancer, responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing, we identify a TAM_C3 cluster, expressing genes implicated in anti-inflammatory activity, that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity, respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus, targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies.

Experimental Approaches

For MDST assays, female FVB mice at 6 weeks of age were purchased from the Jackson Laboratory. Animal experiments were performed in the AALAC approved Animal Facility, Knight cancer Institute, Oregon Health & Science University under IACUC protocol: Combination Therapy Targets Adaptive Resistance in Cancer (TR01_IP00002062). Mice were housed 5 to a cage with ad libitum access to food and water in 20 °C ambient temperatures, 40–50% humidity and a 12-h light/12-h dark cycle. LPA1-T22 and LPA1-T27 have been extensively characterized previously. 25mm3 LPA1-T22 (T22) or LPA1-T127 (T127) tumor chunks were orthotopically transplanted into the mammary fat pad. 10 and 14 days after tumor implantation for T127 and T22 respectively, mice were first assigned with individual numbers and then assigned to treatment groups (n > 5) using an online random number generator. For co-transplantation models, due to differences in growth rates, T127 tumors were transplanted in the opposite mammary fat pad when the T22 tumor reached 20mm3 (~14 days after T22 transplantation). For short term treatment assays, mice were treated with vehicle or olaparib (50 mg/kg, oral gavage, daily, Selleckchem, #S7110) for 4 or 8 days. Tumors were measured by calipers every 2 or 4 days. Tumor volume was calculated according to a modified ellipsoid formula V = 1/2 × (Length × Width2). Mice were euthanized before reaching maximum tumor size of 2000 mm3 according to IACUC guidelines. Mice were isoflurane anesthetized and tumors were collected freshly for single cell RNA sequencing library construction, or cryopreserved as single cell suspension. MDST tumors were disaggregated by gentleMACS kits (Miltenyi Biotec, #130-096-730), and incubated in TotelseqA hashtag antibodies (Biolegend, #394601, #394603, #394605, #394607, #394609, #394611, #394613, #394615) following the manufactural instructions. Live cells were isolated by EasySep Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, #17899). Single cell suspensions were processed according to 10xGenomics scRNAseq sample preparation protocol (Chromium Single Cell 3’ v3.1 Reagent Kit, 10xGenomics). Briefly, single cell suspensions with 2×106 live cells were incubated with 100 μL antibody based hashtag oligos (10ug/ul) for 30 min on ice. After washing 3 times with 3 ml PBS with 10% FBS, 5×105 live hashtagged cells were pooled together followed with dead cell removal column (STEMCELL, 17899) to enrich live cells. Sequencing was done by Novaseq 6000. 10000 cells were targeted for each sample.

scRNA-seq by 10x genomics of two mouse-derived syngeneic transplant (LPA1-T22, and LPA1-T127) single strain transplanted or co-transplanted models treated with olaparib for 4 days, 8 days and 20 days. For co-transplanted models, T22 tumor chunks were transplanted in the opposite site mammary pad 14 days prior to T127 tumors as demonstration in Figure 1A. For short term treatment assays, mice were treated with vehicle or olaparib (50mg/kg, oral galvage, daily, Selleckchem, #S7110) for 4 days, 8 days. For long-term treatment assay mice were treated with vehicle or olaparib (50mg/kg, oral galvage, daily, Selleckchem, #S7110) for 20 days. For MDST assays female FVB mice at 6 weeks of age were purchased from the Jackson Laboratory. Animal experiments were performed by X. Li in Animal Facility, Knight cancer Institute, Oregon Health & Science University. Animal studies were performed according to IACUC approved protocol: Combination Therapy Targets Adaptive Resistance in Cancer (TR01_IP00002062) following AALAC guidelines. Mice were housed 5 to a cage with ad libitum access to food and water in 20℃ ambient temperatures, 40-50% humidity. And 12-hour light/12-hour dark cycle. 25mm3 LPA1-T22 (T22) or LPA1-T127 (T127) tumor chunks were orthotopicly transplanted into the mammary fat pad. 14 or 10 days after tumor transplanted for T22 or T127 respectively, mice were assigned to treatment groups (n>5) based on randomization of tumor size. e dissociation kits (SUM149 xenograft tumors were disaggregated by gentleMACS kits (Miltenyi Biotec, #130-096-730), and incubated in cell multiplexing oligos from 10x Genomics (3' CellPlex Kit Set A 1000261) following the manufactural instructions. Live cells were isolated by EasySep Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, #17899). Single cell suspensions were processed according to 10x Genomics scRNAseq sample preparation protocol (Chromium Single Cell 3’ v3.1 Reagent Kit, 10xGenomics). 5000 cells were targeted for each sample.) Sequenced reads were demultiplexed by Cell Ranger v6.0.0. Sequenced reads were mapped to GRCh38 whole genome with all transcripts by Cell Ranger v6.0.0 with default parameters. Sequenced reads were mapped to GRCh38 whole genome with only exons by velocyto v0.17.

Single Cell RNA Trajectory Analysis Reveals a CD9 Positive Cell State to Contribute to Exit from Stem Cell-like and Embryonic Diapause States and Transit to Drug Resistant States

Bromo- and extra-terminal domain (BET) inhibitors (BETi) have been shown to decrease tumor growth in preclinical models and clinical trials. However, toxicity and rapid emergence of resistance have limited their clinical implementation. To identify state changes underlying acquisition of resistance to the JQ1 BETi, we reanalyzed single-cell RNAseq data from JQ1 sensitive and resistant SUM149 and SUM159 triple-negative breast cancer cell lines. Parental and JQ1-resistant SUM149 and SUM159 exhibited a stem cell-like and embryonic diapause (SCLED) cell state as well as a transitional cell state between the SCLED state that is present in both treatment naïve and JQ1 treated cells, and a number of JQ1 resistant cell states. A transitional cell state transcriptional signature but not a SCLED state transcriptional signature predicted worsened outcomes in basal-like breast cancer patients suggesting that transit from the SCLED state to drug-resistant states contributes to patient outcomes. Entry of SUM149 and SUM159 into the transitional cell state was characterized by elevated expression of the CD9 tetraspanin. Knockdown or inhibition of CD9-sensitized cells to multiple targeted and cytotoxic drugs in vitro. Importantly, CD9 knockdown or blockade sensitized SUM149 to JQ1 in vivo by trapping cells in the SCLED state and limiting transit to resistant cell states. Thus, CD9 appears to be critical for the transition from a SCLED state into treatment-resistant cell states and warrants exploration as a therapeutic target in basal-like breast cancer.

Experimental Approaches

shRNA (shCD9_1 #TRCN0000296954, shCD9_2 #TRCN0000291711, shCD9_3 #TRCN0000296958, shCON #SHC201) were purchased from Sigma Aldrich. Lentivirus were packaged in 293 T cell line by cotransfected into 293 T cells with Lentiviral Packaging Mix (#SHP001, Sigma Aldrich). 72 h after co-transfection, 293 T medium with packaged lentivirus was harvested. SUM149, MDAMB231 and MDAMB468 cells were transfected with 293 T medium containing lentivirus by spinning (1000 × g) for 2 h at 25 °C. Cells were selected in puromycin. SUM149 transfected with shCD9_3 which targets the CD9 3’ untranslated region (UTR) was transfected with CD9-mGFP (0.5 ng/μL, Addgene, #182864) that lacks the CD9 3’ untranslated region to rescue CD9 expression in SUM149 CD9 knockdown cells. scRNA-seq by 10x genomics of shCD9 or shCON SUM149 tumors growing in NSG mouse after 30 days of 25mg/kg/day JQ1 treatment. For xenograft assays 6 weeks old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were purchased from Jackson Laboratory. Tumors were induced by unilateral orthotopic mammary fat pad injection of 2*106 SUM149 cells transfected with shCON or shCD9 lentivirus and suspended in 50 μL of culture medium/Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free (Corning, CLS356231) in a 1:1 ratio. After 14 days, mice were randomized to treatment groups (n=8) based on tumor size. tumor was harvested from NSG mouse mamary pad. Tumor tissue was dissociated with gentleMACS tissue dissociation kits (SUM149 xenograft tumors were disaggregated by gentleMACS kits (Miltenyi Biotec, #130-096-730), and incubated in TotelseqA hashtag antibodies (Biolegend, #394601, #394603, #394605, #394607, #394609, #394611, #394613, #394615) following the manufactural instructions. Live cells were isolated by EasySep Dead Cell Removal (Annexin V) Kit (STEMCELL Technologies, #17899). Single cell suspensions were processed according to 10xGenomics scRNAseq sample preparation protocol (Chromium Single Cell 3’ v3.1 Reagent Kit, 10xGenomics). 1000 cells were targeted for each sample.) RNA libraries were prepared for sequensing using standard library construction protocol by 10x Genomics. Sequenced reads were demultiplexed by Cell Ranger v6.0.0. Sequenced reads were mapped to GRCh38 whole genome with all transcripts by Cell Ranger v6.0.0 with default parameters. Sequenced reads were mapped to GRCh38 whole genome with only exons by velocyto v0.17.