Minimal Xenograft Tumor Growth Variability in Nude Mice Infected with Corynebacterium bovis as a Single Pathogenic Agent
Reduced xenograft engraftment and delayed tumor growth in immunocompromised mice have been associated with infection by Corynebacterium bovis, a highly contagious bacterium that causes sporadic clinical disease and chronic shedding. To learn what effect C. bovis infection may have on the reproducibility of preclinical cancer studies, subcutaneous tumors were monitored in male and female nude (Foxn1nu) mice topically infected with C. bovis 2 to 3 weeks prior to cell injection (chronically infected) or at the time of cell injection (acutely infected) and compared with tumors in uninfected control mice. There were largely no significant differences in tumor engraftment rates, final tumor weights, or final tumor volumes between infection groups for any of the 3 established human cell lines; that is, SJSA-1, an osteosarcoma; HT-29, a colorectal adenocarcinoma; and A549, a lung carcinoma. However, HT-29–engrafted tumors grew significantly more slowly in acutely infected males than in the chronically infected or uninfected males, although no difference was present between the HT-29–engrafted female groups. No epidermal hyperplasia or hyperkeratosis was present in either of the infected groups, compared with the controls, which paralleled the absence of scaly skin clinically across all groups. In summary, our results suggest that C. bovis as a single pathogenic agent may have a limited effect on xenograft engraftment and growth. Investigators using different strains of immunocompromised mice, different cell lines, or mice housed in facilities with different health status from that described in the present study but endemic for C. bovis should perform pilot studies to determine whether tumor engraftment and/or growth is affected by infection.
Experimental design. Upon arrival, 4- to 5-wk-old mice were tested by aerobic dermal culture and fecal qPCR. For each cell line, 3 groups consisting of 5 males and 5 females were created (n = 30 total): 1) chronic infection; 2) acute infection (infection groups had a minimum of 7.5 × 105 cfu applied topically); and 3) sham infection control (media only) following the timeline depicted. All 3 treatment groups were injected with tumor cells on the same day at 8 wk of age with aliquots of tumor cells expanded at the same time. Fecal qPCR testing occurred 7 d after inoculations, on the day of injection, and at the end of the study with a second dermal culture. Upon completion of each experiment, 3 to 4 wk after injection, tumors were collected and weighed, and an intrascapular skin sample was collected and formalin-fixed. Skin samples were examined microscopically and scored.
Whole-genome sequencing of Corynebacterium bovis strains. Fields where data are not available are noted as N/A. (A) Average nucleotide identity percent similarity matrix; values ≥95% indicate that genomes belong to the same species. Corynebacterium bovis strains isolated from rodents (for example, mouse and rat) formed a distinct phylogenetic clade, which included Corynebacterium 1810230001 SH1 (inoculum strain) and C. bovis 2305220054 (output strain). Corynebacterium 1810230001 SH1 and C. bovis 2305220054 share >99.99% similarity, indicating they are likely identical strains. (B) Whole-genome phylogenetic tree built from core genes. Corynebacterium bovis strains isolated from rodents (for example, mouse, and rat) formed a distinct phylogenetic clade, which includes Corynebacterium 1810230001 SH1 and C. bovis 2305220054 (highlighted in yellow). Corynebacterium 1810230001 SH1 and C. bovis 2305220054 are most phylogenetically similar with each other since they are closely located on the same branch in the rodent clade.
Tumor growth parameters of 3 different commonly used established human cancer cell lines (SJSA-1, HT-29, and A549) subcutaneously engrafted into the flanks of male and female nude mice in 1 of 3 experimental groups: 1) acutely infected with Corynebacterium bovis on the day of injection (‘acute’; purple); 2) chronically infected with C. bovis 2 to 3 wk prior to injection (‘chronic’; pink); 3) uninfected controls (blue). (A) Tumor growth rates plotted as mean volume ± SEM from day of injection until tumor collection (n = 5 per group). Rates only differed in the HT-29–injected males (two-way ANOVA, treatment × time; *, P = 0.0008); the acutely infected mice had significantly less growth by day 21 postinjection than both the control and chronically infected mice (Tukey's honestly significant difference test, P < 0.0001). (B) Final tumor weights as a scatter plot; horizontal lines represent the mean. No significant differences were found between groups.
Examples of expected background findings in nude (Foxn1nu) mice; representative images are shown but findings were similar across all tumor cohorts and between both sexes. Histologic hyperplasia and inflammation scores were not significantly elevated in the acute and chronic infection groups, compared with the controls, and appeared similar to these representative images. Corynebacterium bovis was not conclusively identified in sections from the experimentally infected groups, although infection was confirmed via molecular testing and dermal aerobic culture and isolation. (A) Twisting/coiling/fracture of the hair shafts and follicular keratosis (red arrows) in a SJSA-1–engrafted control male. Hematoxylin and eosin stain; original magnification, ×10. (B) Twisting/coiling/fracture of the hair shafts and follicular keratosis (red arrows) along with dermal fibrosis (yellow asterisks) in a HT-29–engrafted control male. Hematoxylin and eosin stain; original magnification, ×10. (C) Epidermal and infundibular hyperplasia (red asterisk) and folliculitis (yellow asterisk) in a A549-engrafted control female. Hematoxylin and eosin stain; original magnification, ×10. (D) Mixed Gram-positive and Gram-negative bacteria within the infundibulum (red arrows) in a A549-engrafted control female (different from female in C). Gram stain; original magnification, ×40.
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