mRNA expression levels of MDR-1 genes.

A. mRNA expression levels of ABCB1 was upregulated in dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C relative to DMSO control 3D-HT-29 spheroid sample. The t-test was used for a statistical test. B. ABCG2 expression levels were upregulated in irinotecan-resistant 3D-HCT-116 spheroid replicates A, B and C relative to DMSO control 3D-HCT-116 spheroids. The t-test was used for a statistical test. Error bars represent mean ± SD. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.</p

3D-HT-29 barcode frequencies.

Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.</div

Experimental design for cellular barcoding of 3D spheroids and their establishment.

A. Schematic of an experimental design. B. Bright field microscope image of 3D-HT-29 spheroids and C. 3D-HTC-116 spheroids. D. The cell viability assay for determining the dabrafenib-dependent sensitives of 3D-HT-29 spheroids and E. irinotecan-dependent sensitivities of 3D-HCT-116 spheroids. Error bars represent mean ± SD.</p

3D-HT-29 common single nucleotide variations.

Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.</div

Validation of copy number alterations and detection of somatic single nucleotide variations in dabrafenib-resistant 3D-HT-29 spheroids.

A. mRNA expression levels of CCNE1, NSD3 and RNF6 genes were upregulated in dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C relative to DMSO control 3D-HT-29 spheroid sample. The t-test was used for a statistical test. B. Variant Allele Frequency (VAF) analysis of dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C found enriched SNVs in FAM98A, RRP15, USP12 and FOXR2 genes relative to initial 3D-HT-29 spheroids. Error bars represent mean ± SD. * = p<0.05, *** = p<0.001, **** = p<0.0001.</p

Establishment of drug resistant derivatives of 3D spheroids.

A. The cell viability assay for the confirmation of dabrafenib resistance in dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C in comparison to DMSO treated spheroids. B. IC50 values of dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C and DMSO control spheroids are presented using bar chart. The one-way ANOVA test was used for a statistical test. C. Brightfield microscope images of dabrafenib-resistant 3D-HT-29 spheroid replicates A, B and C and DMSO control spheroids. D. Dose response analysis for the validation of irinotecan resistance in irinotecan resistant 3D-HCT-116 spheroid replicates A, B and C in comparison to DMSO treated spheroids. E. IC50 values of irinotecan-resistant 3D-HCT-116 spheroid replicates A, B and C in comparison to DMSO treated spheroids were presented using bar chart. The one-way ANOVA test was used for a statistical test. F. Bright field microscope images of irinotecan-resistant 3D-HCT-116 spheroid replicates A, B and C and DMSO treated spheroids are shown. Error bars represent mean ± SD. * = p<0.05, ** = p<0.01.</p

3D-HCT-116 CNV results.

Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.</div

Barcode frequency measurements for the assessment of evolutionary dynamics of drug resistance in 3D spheroids.

A. Barcode frequency measurements of 3D-HT-29 spheroids namely, initial, DMSO control, dabrafenib-resistant replicates A, B, and C. B. Barcode frequency distributions of initial, DMSO control, irinotecan-resistant replicates A, B, and C 3D-HCT-116 spheroids. Barcodes with positive growth rates are classified as pre-existing (amber colour) or de novo (olive green colour) and negative growth rate as sensitive (silver grey colour).</p

3D-HCT-116 barcode frequencies.

Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.</div

3D-HT-29 CNV results.

Complex evolutionary dynamics governing the drug resistance is one of the major challenges in cancer treatment. Understanding these mechanisms requires a sequencing technology with higher resolution to delineate whether pre-existing or de novo drug mechanisms are behind the drug resistance. Combining this technology with clinically very relevant model system, namely 3D spheroids, better mimicking tumorigenesis and drug resistance have so far been lacking. Thus, we sought to establish dabrafenib and irinotecan resistant derivatives of barcoded 3D spheroids with the ultimate aim to quantify the selection-induced clonal dynamics and identify the genomic determinants in this model system. We found that dabrafenib and irinotecan induced drug resistance in 3D-HT-29 and 3D-HCT-116 spheroids are mediated by pre-existing and de novo resistant barcodes, indicating the presence of polyclonal drug resistance in this system. Moreover, whole-exome sequencing analysis found chromosomal gains and mutations associated with dabrafenib and irinotecan resistance in 3D-HT-29 and 3D-HCT-116 spheroids. Last, we show that dabrafenib and irinotecan resistance are also mediated by multiple drug resistance by detection of upregulation of the drug efflux pumps, ABCB1 and ABCG2, in our spheroid model system. Overall, we present the quantification of drug resistance and evolutionary dynamics in spheroids for the first time using cellular barcoding technology and the underlying genomic determinants of the drug resistance in our model system.</div