Dra00b

THP-1 monocyte cells (tib-202tm; ATCC) were cultured in RPMI-1640 medium (12633012; Thermo Fischer) with 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum (FBS). THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce macrophage phenotype as assessed by adherence to tissue culture flask and expression of CD11b integrin (Figure S2). For co-culture, we followed an established protocol^{16 (link),22 (link)}. Briefly, ABCB5+ DSCs or donor-matched ABCB5− fractions were plated at 2x10⁴ cells per well in 24-well plates in 0.5 ml Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS, 100 U/ml penicillin/streptomycin and 2 mM L-glutamine. After 24 hours, THP-1 derived macrophages were seeded on top or in transwell inserts (CLS3464; Corning) at 1×10⁵ cells per well. Co-cultures were incubated with 50 U/ml recombinant human interferon-gamma (IFN-γ) (285-IF-100; R&D Systems) for 24 hours and then stimulated with 20 ng/ml LPS (L3755; Sigma-Aldrich) and 50 U/ml IFN-γ for another 24-hour period before supernatants were harvested and analyzed by enzyme linked immunosorbent assay (ELISA) for IL-1RA (DRA00B; R&D Systems). IL-1RA levels were analyzed between conditions with unpaired t-tests.

Blood samples were typically drawn immediately after completion of assessments. Most participants were assessed between 12:00 and 6:00 P.M, except for ten participants who were assessed between 10:00 A.M. and 12:00 P.M. Blood samples were not drawn for three participants due to time constraints in the participant’s schedule. Blood was immediately spun down at 3,000 × g for 10 minutes and plasma was frozen at −80 °C until batch-wise analyses of inflammatory markers previously associated with cancer-related fatigue in both patients and survivors (Saligan and Kim, 2012 (link); Xiao et al., 2017 ).
Plasma levels of IL-6, sIL-6r, TNF-α, sTNFRII, and IL-1ra were determined with enzyme linked immunosorbent assays (ELISAs) (R&D systems HS600B, DR600, HSTA00D, DRT200, and DRA00B, respectively). Mean minimum detectable levels were 0.039 pg/ml for IL-6, 6.5 pg/ml for sIL-6r, 0.106 pg/ml for TNF-α, 0.6 pg/ml for STNFRII, and 6.3 pg/ml for IL-1ra. One TNF-α sample that was below detection level was set at the mean minimum detectable dose. C-reactive protein (CRP) levels were determined by the MD Anderson Cancer Center core clinical laboratory using high sensitivity chemiluminescent immunometric assay run on a Siemen Immulite XPi. Standard range was 0.2 – 100 pg/mL. One sample exceeded the maximum range for CRP and was repeated at 1:10 dilution.

In vivo release profiles for both IL-1Ra and sTNFRII were obtained using serial collections of serum from mice. Blood was collected at day 1, 5, 14, 21, 28, 42 and 56 following fracture. All serum samples from a single group and collection day were pooled (n = 6-11 per group per time point) in order to obtain sufficient fluid volumes for detection of IL-1Ra (minimum100 μL of sample) or sTNFRII (minimum 20 μL of sample) using commercially available human ELISA kits (DRA00B and DRT200, R&D Systems). For statistical purposes, a value of ½ the lowest level of detection (LLOD) was used for any value that was below the level of detection (IL1-Ra: 3.1 pg-mL and sTNFRII: 0.3 pg-mL). The reported mean intra- and inter-assay coefficients of variation for IL-1Ra are 5.3 % and 8.6 %, respectively. The reported mean intra- and inter-assay coefficients of variation for sTNFRII are 3.5 % and 4.1 %, respectively.