Personalized chemotherapy profiling using cancer cell lines from selectable mice

Abstract

Purpose: High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult because contaminating stromal cells overgrow the malignant cells.

Experimental design: We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts.

Results: Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified 77 U.S. Food and Drug Administration (FDA)-approved drugs with activity, and two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs.

Conclusion: Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice.

Fig. 1. Athymic hprt-null mice

A. Third cross Foxn1^nu/nu mice. The nude hprt-null mouse (II) and heterozygous littermate controls (I, III, IV) show similar phenotypic characteristics in size, hair growth cycles, and skin pigmentation. B. hprt genotyping: hprt PCR products are separated on a 1% agarose gel. Wild type (+) and null (Δ) alleles are assayed in separate PCR reactions. Wild type (WT) mice result in a 746 bp band in the wild type reaction, and only the control 500 bp band in the Δ reaction. Homozygous null females (II, female Null) and hemizygous null males (male Null) result in a 628 bp band in the Δ reaction, but no band in the wild type reaction. Heterozygous females (I, III, IV, HET) result in a 746 bp band in the wild type reaction, and a 628 bp band in the Δ reaction. Note, a 500 bp control band is in the Δ reaction in all lanes. C. Tail cell phenotyping. Homozygous hprt-null (II) and heterozygous (III) tail snips were harvested from female mice, trypsinized and plated in the presence of HAT or 6TG containing media. Brightfield microscopy of crystal violet stained cells.

Fig. 2. Rapid pancreatic cancer cell line production

Samples explanted from Panc410 tumors grown in a nude hprt-null mouse (A–F) in the presence (A–C) or absence (D–F) of HAT media, and photographed using phase microscopy at T=0, 10 and 19 days in culture. Note that after 19 days in the absence of HAT media (F), fibroblasts have overgrown the culture.

Fig. 3. Chemosensitivity of a low-passage familial pancreatic cancer from surgery

Histogram of the number of drugs (frequency) as a function of % growth inhibition (A). Curve-fitting of Gaussian distribution onto the histogram (black line) distinguishes the distribution of drugs with little or no activity from those which demonstrate some level of activity above this distribution. Arrowhead indicates 3 standard deviations (+3SD) above the mean. Arrows indicate the % growth inhibition for nogalamycin (N) and digitoxin (D). Cell line specific sensitivity of nogalamycin and digitoxin in the cell lines Panc502, Panc486 and Panc410 (B). Values shown are the mean IC₅₀ values of 3 replicates and error bars are the 95% confidence intervals. In vivo growth curves of subcutaneous mouse xenografted tumors raised from the Panc410 and Panc502 cell lines after treatment with nogalamycin, digitoxin or control (C). Control (empty ovals), nogalamycin 0.2 mg/kg (empty squares), nogalamycin 1.0 mg/kg (filled squares), digitoxin 0.4 mg/kg (empty triangles), digitoxin 2.0 mg (filled triangles). Normalized weight of tumors explanted from mice after 30 days of treatment (D). Normalized tumor weight of Panc410 and Panc502 in white columns or grey columns, respectively. Error bars are standard deviations. Fold changes between Panc410 and Panc502 are noted.