A combinatorial genetic strategy for exploring complex genotype–phenotype associations in cancer

Available genetically defined cancer models are limited in genotypic and phenotypic complexity and underrepresent the heterogeneity of human cancer. Here, we describe a combinatorial genetic strategy applied to an organoid transformation assay to rapidly generate diverse, clinically relevant bladder and prostate cancer models. Importantly, the clonal architecture of the resultant tumors can be resolved using single-cell or spatially resolved next-generation sequencing to uncover polygenic drivers of cancer phenotypes.

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Sincerely, Safia Danovi
Editor Nature Genetics Referee expertise: Referee #1: prostate/bladder cancer models Referee #2: cancer models, functional genomics Referee #3: cancer models Reviewers' Comments: Reviewer #1: Remarks to the Author: The study by Li et al claims to investigate cooperative mechanisms in cancer development using a novel bar coding approach.In principal, this should be an interesting application of new technologies.However, the study fails to do so and largely incomplete.
Major concerns include the following: 1.Overall, the work is descriptive, mechanistic insights are mostly lacking, as is the validation to human cancer (mouse models are investigated).2. Overall, the study is vague -the abstract in particular does not actually describe a cancer focus or purpose.
3. The Novelty is limited -how does this study/methodology differ from or advance beyond work done by Owen Witte's lab (which used human not mouse tissues)?4. Related to point #3, the novelty of the bar coding method is unclear as well. 5.A major concern is the overall vagueness of the study and the way it is written.6.Overall, the rationale for focusing this study on bladder and prostate is unclear.
Reviewer #2: Remarks to the Author: The authors describe an innovative approach based on the optimized transduction of mouse primary epithelial cells with barcoded lentiviral libraries of oncogenic events to identify cancer genes that cooperate to transform the target cells into new multifactorial genetic models of cancer.The manuscript is very well written, and the scope and approach are clear, novel and compelling and would be of interest to the cancer community, especially in the field of novel mouse model development.
I do have some minor comments regarding the genetic characterization of the established models and their comparability to their human counterparts that, if addressed, I believe would further improve the relevance of this study.
1.The authors identify sets of cancer genes whose combined alterations result in specific types of cancer histology (e.g.Fgfr3 S243C,116 Ywhaz, Pik3ca E545K, Pparg, and Pvrl4 produce papillary urothelial carcinoma with inverted growth pattern.How frequently do the same gene alterations cooccur in human cancers of the same histology?2. Since combinatorial genetics is described as the major innovation of the study, I was expecting to see single-cell DNA amplicon sequencing results and deconvolution of the oncogenic events associated with each one of the tumors in the FHBT series.Please explain why the data is not available/not presented, and why the study pivoted to focus on transcriptional profiling instead.
3. The conclusion that the FHIB models are relevant because they 'occupy overlapping space' in the PCA plots of the TCGA BLCA cohort and BBN bladder mouse tumors requires some more detailed explanation.It would be significant to comment on the types/frequencies of cancer gene alterations shared between mouse and human cancers of the same histology and/or transcriptional subtypes.

Minor points
4. Please clarify the nature of the different Mission Bio Tapestri custom single cell amplicon panels: the one described in Extended Data Fig. 1c vs. the one used for the detection of the cancer gene LV Barcodes in Fig. 1f?Is the first one just a test panel to confirm that individual BCs can be detected with high resolution in the premixed 3T3 cell populations?How does it relate to the fig.1f panel? 5. Please explain how the histological subtype assignments in Fig. 2e are made with reference to the corresponding histological subtype mixture as reported in Fig. 2a?Is it based on the prevalent subtype?
Reviewer #3: Remarks to the Author: The manuscript submitted authored by by Li, Wong and Sun,.et al., presents a novel methodology for transforming and barcoding mouse primary cells, enabling the generation of bladder and prostate cancer models with clinical relevance when injected into immunodeficient mice.The team has conducted an in-depth investigation into the role of commonly mutated genes observed in human bladder and prostate cancers, with a particular focus on elucidating the specific combination of mutations necessary for tumorigenesis.I find this approach to be exceptionally intelligent and intriguing, and the manuscript effectively outlines the technique employed.However, I believe the team should have no difficulty addressing the following comments, which aim to enhance the clarity and precision of the study.

Major points:
In Extended Data 1a, it would be informative to show all the plots for bladder and prostate tissues.Currently, only the isolation plot for one tissue is shown, while two different tissues were used.To maintain consistency with panel b, where organoids from both tissues are depicted after isolation, it would be convenient to showcase both bladder and prostate isolation plots.
In Extended Data 2c, the relative expression of target genes is displayed by qPCR, but only the downregulated genes using shRNA are presented.It would be valuable to also exhibit the upregulation of those genes that are overexpressed (GOF) to facilitate comparison with the actual expression levels observed in human tumours.
Figure 1d lacks the inclusion or mention of Ctrl grafts in mice using solely embryonic bladder and urogenital mesenchymal cells.It is important to clarify whether this control experiment was performed.
In Figure 1, it would be beneficial to include information regarding the number of mice and tumours obtained, as well as the efficiency of tumour formation.
In Extended Figure 6b, the authors state that their FHBT tumour model exhibits RNA expression patterns resembling those of human cancers from TCGA-BLCA and nitrosamine-induced mouse cancer models.However, the PCA plot demonstrates significant differences between them.This disparity should be addressed and explained more comprehensively.

Minor points:
In Extended Figure 7, tumour cells isolated from tumours were propagated in vitro using organoids to increase cell numbers and analyse mutation burden.However, the authors do not mention the number of passages given, which could potentially impact the mutational landscape.It is crucial to consider that culture conditions might affect mutations within the organoids.
In the text (line 200), there appears to be a typographical error where "urogenital sinus mesenchyme" is duplicated.
In the text (line 227), it would be valuable to provide information on the duration it takes for the tumours to reach 1cm.Additionally, it should be clarified whether this timeframe is dependent on the tumour type and tissue.
Comment 1: Overall, the work is descriptive, mechanistic insights are mostly lacking, as is the validation to human cancer (mouse models are investigated).
Thank you very much for your time and effort in reviewing our manuscript.We have submitted this manuscript as a Brief Communication given our belief that these initial results validate the utility of this novel combinatorial genetics approach to rapidly 1) generate diverse cancer models including clinically relevant bladder cancer and prostate cancer histologies that have not been recapitulated in available genetically engineered mouse models and 2) interrogate genotype-phenotype relationships.We agree that deep mechanistic insights are lacking in this initial report, but additional studies are ongoing and already yielding exciting results that we intend to publish in the future.
Comment 2: Overall, the study is vague -the abstract in particular does not actually describe a cancer focus or purpose.

Thank you for this comment. The abstract of a Brief Communication is limited to three sentences/70 words and we tried our best to succinctly convey the most salient points of the manuscript within this constraint. To further clarify the purpose of the study, we have now significantly amended the first paragraph of the main text in lines 48-69 to elaborate on the cancer focus.
Comment 3: The Novelty is limited -how does this study/methodology differ from or advance beyond work done by Owen Witte's lab (which used human not mouse tissues)?
Thank you for allowing clarification of this point.We have added text to lines 50-56 and 57-64 to describe potential shortcomings of available cancer models and current genetically-defined methodologies employed in functional cancer genomics.We also mention the need for advances in "scale, throughput, and economy" on line 65 relative to established geneticallyengineered mouse models and dissociated-cell tissue recombination/transplantation assays for cancer.In this context, we describe how the methodology described may overcome prior limitations and provide a clearer basis for novelty.

The studies performed by Dr. Witte's laboratory were reliant on a low-throughput approach with parallel testing of combinations of defined sets of genetic insults (i.e., each graft receives either genes A+B or A+C or B+C or A+B+C+D).
A key advancement of our study is the ability to introduce-at high efficiency and throughput (as shown in Fig. 1b and 1c)-many combinations of gain-or loss-of-function genetic events simultaneously from a barcoded lentiviral library within each graft (i.e., genes A-Z where individual cells within the graft receive diverse gene combinations A+B+C, B+E+G+H+Z, A+C+R+X+Y+Z, etc.).Further, we have implemented custom, highly sensitive, targeted single-cell DNA amplicon sequencing in a novel manner to enable massively parallel deconvolution of multiple barcoded lentiviruses integrated into individual cells within a tumor.This allows us to track the clonal architecture of the tumor and associate genetic interactions that may have promoted tumorigenesis and specific tumor phenotypes.
We have added language to the manuscript in lines 183-188 to further highlight the value of this methodology in our proof-of-concept studies: "We leveraged this strategy to develop a series of mouse bladder cancers that recapitulate the phenotypic diversity of human bladder cancer and a mouse prostate cancer with pleomorphic giant cell carcinoma, representing cancer subtypes that have not previously been modeled in a genetically-defined fashion.Importantly, single-cell LV barcode deconvolution associated mutant active Pparg with luminal papillary differentiation of urothelial carcinoma and loss of Kmt2c with pleomorphic giant cell carcinoma in prostate cancer." Comment 4: Related to point #3, the novelty of the bar-coding method is unclear as well.
We have addressed the novelty of the barcoding method and single-cell DNA amplicon sequencing strategy in our response to Comment 3. Comment 5: A major concern is the overall vagueness of the study and the way it is written.
We have made changes to the manuscript text as described in responses to Comments #2 and #3 to better convey how this study represents a scientific advance and the relative importance of our results.
Comment 6: Overall, the rationale for focusing this study on bladder and prostate is unclear.
We focused on bladder and prostate cancer given our prior experience isolating primary mouse epithelial cells from these tissues and our cancer-specific expertise in this area.Specifically related to bladder cancer, available genetically-engineered mouse models have poorly reflected the diversity of the human disease and so our intention was to seize this as an opportunity to show that we could recapitulate diverse bladder cancer phenotypes including variant histologies by employing this combinatorial genetic approach.While the manuscript focuses on bladder and prostate cancer for proof-of-concept studies, we believe that this approach may be applicable to and have broad utility for the study of many cancer types and other polygenic diseases.

Reviewer #2 (Remarks to the Author):
The authors describe an innovative approach based on the optimized transduction of mouse primary epithelial cells with barcoded lentiviral libraries of oncogenic events to identify cancer genes that cooperate to transform the target cells into new multifactorial genetic models of cancer.
The manuscript is very well written, and the scope and approach are clear, novel and compelling and would be of interest to the cancer community, especially in the field of novel mouse model development.
I do have some minor comments regarding the genetic characterization of the established models and their comparability to their human counterparts that, if addressed, I believe would further improve the relevance of this study.
Comment 1: The authors identify sets of cancer genes whose combined alterations result in specific types of cancer histology (e.g.Fgfr3 S243C, Ywhaz, Pik3ca E545K, Pparg, and Pvrl4 produce papillary urothelial carcinoma with inverted growth pattern.How frequently do the same gene alterations co-occur in human cancers of the same histology? Thank you very much for your careful consideration of our manuscript.We agree that it is important to include information regarding the co-occurrence of these alterations in human bladder cancer.We have now added Extended Data Fig. 5 (shown below) which is an Oncoprint from cBioPortal showing the frequencies and types of gene alterations associated with human muscle-invasive bladder cancer from the TCGA-BLCA cohort.We focus specifically on the gain-and loss-of-function alterations encoded in our mBU-LVp including Pparg, Pvrl4, Ywhaz, Pik3ca, and Fgfr3.While the types of histologies are not annotated in this dataset, it is evident that these gene alterations co-occur.As we have described in the manuscript, FGFR3 activating mutations have previously been shown to be highly enriched in papillary urothelial carcinomas.
Comment 2: Since combinatorial genetics is described as the major innovation of the study, I was expecting to see single-cell DNA amplicon sequencing results and deconvolution of the oncogenic events associated with each one of the tumors in the FHBT series.Please explain why the data is not available/not presented, and why the study pivoted to focus on transcriptional profiling instead.
We agree that it is important to deconvolute the oncogenic events associated with each of the tumors in the FHBT series.In the manuscript, we show deconvolution of one of the FHBT tumors and functional validation of the role of Pparg in driving luminal papillary urothelial carcinoma.We also associate loss of Kmt2c with pleomorphic giant cell carcinoma in a prostate tumor.However, we are actively deconvoluting all of the bladder and prostate tumors and performing functional studies to understand the key genetic interactions that may be critical in driving each cancer phenotype.As you can imagine, this work will take some time and is already yielding exciting results that we intend to publish in the future.Our intention was to submit this initial manuscript as a proof-of-concept of the technology and its potential utility.We focused on transcriptional profiling for more robust phenotypic validation of the clinical relevance of the FHBT models.
Comment 3: The conclusion that the FHIB models are relevant because they 'occupy overlapping space' in the PCA plots of the TCGA BLCA cohort and BBN bladder mouse tumors requires some more detailed explanation.It would be significant to comment on the types/frequencies of cancer gene alterations shared between mouse and human cancers of the same histology and/or transcriptional subtypes.
We have updated the PCA projection plots in Fig. 2i and Extended Data Fig. 7b (shown below) after incorporating batch correction using ComBat-seq between the datasets.We have now included 90% confidence ellipses for each histology and/or consensus classification to enhance the visual interpretation of the results.We have also added clarification in the text on lines 161-163: "…occupy overlapping space based on histologic classification, indicating that the transcriptional features with the greatest variance between tumor subtypes are also conserved with FHBT models." As mentioned, we have added an Oncoprint to Extended Data Fig. 5 to show specifically genes included in our mBU-LVp and their alteration frequencies in human muscle-invasive bladder cancer from the TCGA-BLCA cohort.Unfortunately, the overwhelming majority of TCGA-BLCA cases are annotated as "urothelial carcinoma" and there are only a handful of variant histologic subtypes within the cohort.Pathologists often do not note variant histologic subtypes of bladder cancer unless it is the dominant cancer histology.Thus, the small number of such annotated cases with cancer genome sequencing makes it currently difficult to answer the frequencies of gene alterations shared between mouse FHBT models and human bladder cancers of the same histologic subtypes.

Minor points
Comment 4: Please clarify the nature of the different Mission Bio Tapestri custom single cell amplicon panels: the one described in Extended Data Fig. 1c vs. the one used for the detection of the cancer gene LV Barcodes in Fig. 1f?Is the first one just a test panel to confirm that individual BCs can be detected with high resolution in the premixed 3T3 cell populations?How does it relate to the fig.1f panel?We apologize that this was not clear.Extended Data Fig. 1c describes the Mission Bio Tapestri custom single-cell amplicon sequencing panel that was applied in the experiments shown in Fig. 1f and Extended Data Fig. 1d. The experimental data in Extended Data Fig. 1d is shown to confirm the ability of the panel to accurately and sensitively deconvolute the clonal architecture of a well-defined set of 3T3 cells with mixtures of clones harboring different combinations of lentiviral barcodes.After validating this technology, we then applied the custom single-cell amplicon sequencing panel to our dissociated tumor as shown in Fig. 1f.
Comment 5: Please explain how the histological subtype assignments in Fig. 2e are made with reference to the corresponding histological subtype mixture as reported in Fig. 2a?Is it based on the prevalent subtype?
Yes, the histological subtype assignment in Fig. 2e was made based on the prevalent subtype.

Reviewer #3 (Remarks to the Author):
The manuscript submitted authored by by Li, Wong and Sun,.et al., presents a novel methodology for transforming and barcoding mouse primary cells, enabling the generation of bladder and prostate cancer models with clinical relevance when injected into immunodeficient mice.The team has conducted an in-depth investigation into the role of commonly mutated genes observed in human bladder and prostate cancers, with a particular focus on elucidating the specific combination of mutations necessary for tumorigenesis.I find this approach to be exceptionally intelligent and intriguing, and the manuscript effectively outlines the technique employed.However, I believe the team should have no difficulty addressing the following comments, which aim to enhance the clarity and precision of the study.

Major points:
Comment 1：In Extended Data 1a, it would be informative to show all the plots for bladder and prostate tissues.Currently, only the isolation plot for one tissue is shown, while two different tissues were used.To maintain consistency with panel b, where organoids from both tissues are depicted after isolation, it would be convenient to showcase both bladder and prostate isolation plots.

Thank you very much for these excellent comments. As suggested, we have now included representative bladder (top) and prostate (bottom) isolation plots in Extended Data Fig. 1a (shown below).
Comment 2：In Extended Data 2c, the relative expression of target genes is displayed by qPCR, but only the downregulated genes using shRNA are presented.It would be valuable to also exhibit the upregulation of those genes that are overexpressed (GOF) to facilitate comparison with the actual expression levels observed in human tumours.
Thank you for this suggestion.We have validated the upregulation of the overexpressed (gainof-function) genes in 3T3 cells by qPCR.This data is now plotted in Extended Data Fig. 2d (shown below).We have also added the identities of the primers used for these studies to Extended Data Table 3.
Comment 3：Figure 1d lacks the inclusion or mention of Ctrl grafts in mice using solely embryonic bladder and urogenital mesenchymal cells.It is important to clarify whether this control experiment was performed.
We appreciate this comment.We did add this key control in our experiments and none of these grafts formed tumors.We have now clarified this in line 114-115 of the text: "No tumors were appreciable from control grafts of untransduced mBU or mPE cells recombined with EBLM or UGSM."

Comment 4：In
Figure 1, it would be beneficial to include information regarding the number of mice and tumours obtained, as well as the efficiency of tumour formation.
We agree that this is beneficial and important information.We have now included Extended Data Table 2 (shown below) that summarizes the efficiency of tumor formation.We have added a description of this data to the text on lines 115-117: "The efficiency of tumor formation (tumors formed per graft inoculated) was 80% (16 of 20) for mBU cells infected with

Tissues Number of mice inoculated Tumor incidence (%) Average time to 1 cm tumor diameter (months)
Bladder 20 80% 4.2 Prostate 47 38% 8.9 Comment 5：In Extended Figure 6b, the authors state that their FHBT tumour model exhibits RNA expression patterns resembling those of human cancers from TCGA-BLCA and nitrosamine-induced mouse cancer models.However, the PCA plot demonstrates significant differences between them.This disparity should be addressed and explained more comprehensively.
We appreciate this comment.We did not previously apply batch correction but after using ComBat-seq, we find that the PCA projections now show greater similarity between tumor subtypes comparing FHBT to TCGA-BLCA and BBN tumors.Our response to Comment 3 from Reviewer 2 shows these new plots and our additional description of these results.

Minor points:
Comment 6：In Extended Figure 7, tumour cells isolated from tumours were propagated in vitro using organoids to increase cell numbers and analyse mutation burden.However, the authors do not mention the number of passages given, which could potentially impact the mutational landscape.It is crucial to consider that culture conditions might affect mutations within the organoids.
We propagated the cells for one passage to increase cell numbers for further downstream analysis.This information has been provided in the revised manuscript on line 172.
Comment 7：In the text (line 200), there appears to be a typographical error where "urogenital sinus mesenchyme" is duplicated.
Thank you for catching this typographical error.This has been corrected on line 233 to replace one of the "urogenital sinus mesenchyme" with "embryonic bladder mesenchyme." Comment 8：In the text (line 227), it would be valuable to provide information on the duration it takes for the tumours to reach 1cm.Additionally, it should be clarified whether this timeframe is dependent on the tumour type and tissue.
We have now included Extended Data Thank you for submitting your revised manuscript "Combinatorial genetic strategy accelerates the discovery of cancer genotype-phenotype associations" (NG-BC62414R).It has now been seen by the original referees and their comments are below.The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Genetics, pending minor revisions to comply with our editorial and formatting guidelines.
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