Enhancement of cisplatin-based TACE by a hemoglobin-based oxygen carrier in an orthotopic rat HCC model

Figures & data

Figure 1. Decreased cisplatin-sensitivity of McA-RH7777 cells under hypoxia. Rat hepatoma cell line McA-RH7777 was cultured in either normoxic (Nx) or hypoxic (Hx) condition (1%) in the presence of cisplatin for 24 h or 48 h. Cell survival was then assessed by MTT assay. *p < 0.05.

Figure 2. Attenuation of hypoxia by OC89 in tumor tissues. The rats were divided into two groups, being given either Ringer's buffer or oxygen carrier via tail vein injection. pO₂ readings were measured both before and 1 h after interference. (a) Improved oxygenation by OC89. The data were shown as mean ± SD. * p < 0.05. (b) Shift of pO₂ fraction from low (0–10 mmHg) to high readings (15–25 mmHg) after OC89 delivery. (c) Expression of hypoxia marker pimonidazole after interference. Immunostaining showed less binding to pimonidazole (lower panel) in tumor tissue after delivery of OC89 (Bar = 100 um).

Table I. pO₂ readings (mmHg) inside tumor tissue before and after OC89 delivery.

	Pre-intervention			Post-intervention
	Ringer's buffer group	OC89 group	P value	Ringer's buffer group	OC89 group	P value
Range	− 0.7–30.9	− 0.6–29.8		− 0.7–26.8	1.7–51.3
Mean^¶	8.1 (± 7.7)	9.2 (± 7.0)	0.368	7.4 (± 6.5)	23.3 (± 12.6)	0.000
Median^§	6.8	9.4	0.231	6.7	22.7	0.000

Rats were divided into two groups (three in each group) and either Ringer's buffer or OC89 was given. pO₂ readings before any interference in the two groups was comparable while a significant upregulation of pO₂ readings in tumor tissues was observed 1 h after intervention with OC89.

^¶T-Test; ^§Mann–Whitney U test.

Table II. Tumor volume changes in different therapeutic regimens.

		Tumor volume (mm^3)
Groups	Regimens	Pre-treatment	Post-treatment	P value
A	Cisplatin(L) + Ringer(L)*	790 ± 417	4910 ± 1201	0.65
	Cisplatin(L) + OC89(L)	810 ± 403	5520 ± 1738
B	Cisplatin(L) + Ringer(H)^†	790 ± 410	6700 ± 989	0.10
	Cisplatin(L)+ OC89(H)	680 ± 257	3000 ± 1470
C	Cisplatin(H) + Ringer(L)	750 ± 280	1540 ± 998	0.24
	Cisplatin(H) + OC89(L)	500 ± 359	680 ± 570
D	Cisplatin(H)+ Ringer(H)	620 ± 251	1980 ± 1020	0.03
	Cisplatin(H) + OC89(H)	570 ± 294	410 ± 187

Four regimens were tested and the changes of tumor volumes before (Day 0) and after therapy (Day 21) were compared. Cisplatin was given at 1 mg/kg (low dose) or 3 mg/kg (high dose); OC89 was given at 0.2 g/kg (low dose) or 0.4 g/kg (high dose). Ringer's buffer of the same volume as OC89 was delivered accordingly. Pretreatment tumor volume was estimated by ab²/2; posttreatment tumor volume was calculated by πcde/6. The data were shown as mean ± SD and analyzed with T test.

*L: low dose; ^†H: high dose.

Figure 3. Enhanced therapeutic effect in TACE by OC89. (a) MRI or xenogen images in rats before or after TACE with or without OC89 delivery. The images were taken at Day 0 (left panel) or 21 days after TACE (middle and right panel). (b) Macro- and microscopic evaluation of tumors 21 days after TACE. In OC89-treated rat, the implanted tumor was replaced by granuloma (lower right, arrow) and proliferative fibrous tissue; while in rats receiving buffer, living tumor cells (lower left, solid arrow) accompanied with partial necrosis (lower left, dashed arrow) were observed. Bar = 20 um. (c) and (d) Altered expression of GRP78 and CHOP in rats at Day 1 after TACE. Bar = 100 um. (e) and (f) Increase of apoptosis (TUNEL assay, Day 1) and inhibition of proliferation (Immunostaining of Ki-67, Day 21) by OC89 in tumor cells after TACE. Bar = 100 um. *p < 0.05.

Figure 4. Differential expression of GRP78 in human TACE-sensitive or resistant HCC. (a) Weak staining of GRP78 in tumor cells of TACE-sensitive HCC and strong positivity in tumor cells of TACE-resistant HCC (Upper panel, Bar = 20 um; lower panel, bar = 100 um). (b) Scoring of GRP78 staining. The scoring was calculated based on both staining intensity and area of positivity.